Cationic steroid antimicrobial compositions and methods of use

ABSTRACT

The invention provides methods for decreasing or inhibiting poxvirus infection or pathogenesis of a cell in vitro, ex vivo or in vivo, a symptom or pathology associated with poxvirus infection or pathogenesis in vitro, ex vivo or in vivo, or an adverse side effect of poxvirus infection or pathogenesis in vitro, ex vivo or in vivo. In one embodiment, a method of the invention includes treating a subject with an invention compound (e.g., cationic steroid antimicrobial or CSA).

RELATED APPLICATIONS

This application claims the benefit of priority of provisionalapplication Ser. No. 60/764,218, filed Feb. 1, 2006, which is expresslyincorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with Government support under grantN01-AI-40029, awarded by the National Institutes of Health. TheGovernment has certain rights to this invention.

TECHNICAL FIELD

The invention relates to methods for decreasing or inhibiting poxvirusinfection or pathogenesis of a cell in vitro, ex vivo or in vivo, asymptom or pathology associated with poxvirus infection or pathogenesisin vitro, ex vivo or in vivo, or an adverse side effect of poxvirusinfection or pathogenesis in vitro, ex vivo or in vivo. In oneembodiment, a method of the invention includes treating a subject withan invention compound (e.g., cationic steroid antimicrobial or CSA).

INTRODUCTION

A smallpox vaccine was the first human vaccine, and vaccinia virus (VV)is considered the most successful human vaccine, bringing about theworldwide eradication of smallpox disease. Mechanisms of adaptive immuneprotection elicited by the smallpox vaccine in humans generally remainunclear.

There is currently greatly renewed interest in smallpox due to thepossible threat of bioterrorism (Henderson et al., Jama 281:2127(1999)). Given this concern, there has been much discussion both aboutthe mechanisms of protection afforded by the smallpox vaccine andpossible development of alternatives to vaccines such as Dryvax®.

SUMMARY

Cationic steroid antimicrobials (CSAs) were developed as functionalmimics of endogenous peptide antibiotics such as LL-37. A series of CSAshave been developed and CSAs are highly active against specificlipid-enveloped viruses including poxvirus (vaccinia). Antiviralactivities of multiple CSAs have been measured, and active and inactiveforms have been identified. In addition, CSAs preferentially associatewith viral envelopes over host cells (keratinocytes).

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing compounds of the invention.

FIG. 2 is a drawing showing compounds CSA-26 and CSA-46.

FIG. 3 is a drawing showing compound 134.

FIG. 4 is a drawing showing compound CSA-10.

FIG. 5 is a dr awing showing compound 140.

FIG. 6 is a drawing showing compound CSA-31.

FIG. 7 is a drawing showing compounds 352-354.

FIG. 8 is a drawing showing compounds 341-343 and 324-327.

FIG. 9 is a drawing showing compounds 358.

FIGS. 10A and 10B are drawings showing various compounds of theinvention (CSAs).

FIG. 11 shows the ability of CSA-8, -13 and -31 to kill vaccinia virus.

FIG. 12 shows Vaccinia virus killing by CSAs determined byquantification of viral RNA.

FIG. 13 shows fluorescence of CSA-59 (450 nm) in the presence of variedamounts of surface area of vaccinia virus and keratinocytes. The numberof PFU of vaccinia and numbers of keratinocytes were varied, and surfaceareas were plotted to account for the large difference in surface areaof the virus and cells. An increase in fluorescence of CSA-59 has beencorrelated to incorporation of CSA in a lipid bilayer.

DETAILED DESCRIPTION

In accordance with the invention, there are provided methods fordecreasing or inhibiting poxvirus infection or pathogenesis of a cell invitro, ex vivo or in vivo, a symptom or pathology associated withpoxvirus infection or pathogenesis in vitro, ex vivo or in vivo, or anadverse side effect of poxvirus infection or pathogenesis in vitro, exvivo or in vivo. In one embodiment, a method of the invention includestreating a subject with an invention compound (e.g., cationic steroidantimicrobial or CSA), wherein the subject is in need of treatment dueto CSA anti-poxvirus activity or function, in order to provide thesubject with a beneficial effect or improvement. In another embodiment,a method of the invention includes providing a subject with protectionagainst a poxvirus infection or pathogenesis by administering acomposition comprising a sufficient amount of CSA to provide the subjectwith protection against a poxvirus infection or pathogenesis. In afurther embodiment, a method of the invention includes treating asubject for poxvirus infection or pathogenesis by administering acomposition comprising a sufficient amount of CSA to treat the subjectfor the poxvirus infection or pathogenesis. In an additional embodiment,a method of the invention includes decreasing susceptibility of asubject to a poxvirus infection or pathogenesis by administering acomposition comprising a sufficient amount of CSA to decreasesusceptibility of the subject to a poxvirus infection or pathogenesis.Methods of the invention include administering CSA prior to,concurrently with, or following contact of the subject with or exposureof the subject to a poxvirus; and administering CSA prior to,concurrently with, or following development of a symptom or pathologyassociated with or caused by poxvirus infection. In various aspects, acompound of the invention (e.g., CSA) is administered prior to(prophylaxis), concurrently with or following infection or exposure ofthe subject (therapeutic) to a poxvirus.

The invention treatment methods therefore include, among other things,therapeutic and prophylactic methods. Subjects can be contacted with,administered ex vivo or in vivo delivered a compound of the invention(e.g., CSA) prior to, concurrently with or following poxvirus exposureor contact, poxvirus infection, or development of a symptom or pathologyassociated with or caused by a poxvirus infection or pathogenesis.

The term “therapeutic” and grammatical variations thereof means thesubject has a poxvirus infection, for example, the subject exhibits oneor more symptoms or pathologies associated with or caused by poxvirusinfection or pathogenesis as set forth herein or known in the art. Theterm “therapeutic” also includes a subject that has been exposed to orcontacted with a poxvirus but may not exhibit one or more symptoms orpathologies associated with or caused by poxvirus infection orpathogenesis, as set forth herein or known in the art.

“Prophylaxis” and grammatical variations thereof refer to contact,administration or in vivo delivery to a subject prior to a known contactwith or exposure to poxvirus. In situations where it is not known if asubject has been contacted with or exposed to poxvirus, contact with,administration or in vivo delivery of a compound to a subject occursprior to manifestation or onset of a symptom associated with or causedby poxvirus infection or pathogenesis. In such a method, the effect ofcontact with, administration or in vivo delivery of a compound of theinvention (e.g., CSA) can be to eliminate, prevent, inhibit, decrease orreduce the probability of or susceptibility towards developing apoxvirus infection or pathogenesis, or a symptom or pathology associatedwith or caused by poxvirus infection or pathogenesis.

As used herein, the term “associated with,” when used in reference tothe relationship between a symptom, pathology or adverse side effect ofvaccination, and a poxvirus, means that the symptom, pathology or sideeffect is caused by poxvirus infection, pathogenesis or vaccination, oris a secondary effect of the poxvirus infection, pathogenesis orvaccination. A symptom, pathology or side effect that is present in asubject may therefore be the direct result of or caused by the poxvirusinfection, pathogenesis or vaccination, or may be due at least in partto the subject reacting or responding to poxvirus infection,pathogenesis or vaccination (e.g., the immunological response). Forexample, a symptom or pathology that occurs during a poxvirus infection,pathogenesis or vaccination may be due in part to an inflammatoryresponse of the subject.

The invention also provides methods for decreasing or preventing anadverse side effect caused by vaccination of a subject with a poxvirus.In one embodiment, a method includes administering a sufficient amountof CSA to the subject to decrease or prevent an adverse side effectcaused by vaccination with a poxvirus. In one aspect, the poxviruscomprises a Vaccinia virus.

In particular embodiments of the compounds and methods of the invention,a CSA is selected from: CSA-7, CSA-8, CSA-10, CSA-11, CSA-13, CSA-15,CSA-17, CSA-21, CSA-25, CSA-26, CSA-31, CSA-46, CSA-54 and CSA-59, asset forth in FIG. 10. In other embodiments, a CSA does not have acharged group at position C24 or a CSA has a hydrophobic moiety atposition C24 (e.g., a lipid). In additional embodiments, a CSA has acharged group at position C7. In further embodiments, a CSA comprises amultimer (e.g., a dimer, trimer, tetramer or higher order polymer). Inyet additional embodiments, a CSA has a shorter tether length betweenthe steroid scaffold and any amine group at positions C3, C7 or C12,relative to the tether length between the steroid scaffold and any aminegroup at positions C3, C7 or C12 of CSA-7, CSA-8, CSA-10, CSA-11,CSA-13, CSA-15, CSA-17, CSA-21, CSA-25, CSA-26, CSA-31, CSA-46, CSA-54or CSA-59, as set forth in FIG. 10.

Methods of treatment include reducing, decreasing, inhibiting,ameliorating or preventing onset, severity, duration, progression,frequency or probability of one or more adverse side effects associatedwith poxvirus vaccination. Non-limiting examples of adverse side affectsassociated with poxvirus vaccination treatable with a compound of theinvention include postvaccinial encephalitis, progressivevaccinia-necrosum, eczema vaccinatum, Vaccinia Keratitis, generalizedvaccinia, myopericarditis, inoculation of other sites, infection ofclose contacts, rash and periocular infection and death.

Methods of the invention, including, for example, prophylactic andtherapeutic treatment methods, as well as methods for decreasing orpreventing an adverse side effect caused by vaccination with or againstpoxvirus, are applicable to poxvirus generally, more specifically, themembers of the family Poxviridae. Poxvirus includes any strain orisolate or subtype or a species of poxvirus, or combination of strainsor isolates or subtypes or species of poxviruses. Particular examplesare infectious or pathogenic viruses. Specific non-limiting examples ofpoxvirus the subject of treatment with an invention compound (e.g., CSA)include, for example, live or attenuated pathogenic and non-pathogenicpoxvirus. Exemplary pathogenic poxvirus include, variola major andvariola minor smallpox virus. Particular non-limiting examples ofvariola major or variola minor smallpox virus include extracellularenveloped virus (EEV), intracellular mature virus (IMV) andcell-associated virus (CEV) forms. Additional exemplary pathogenicpoxvirus include monkeypox, cowpox, Molluscum Contagiosum, camelpox,goatpox, swinepox, sheeppox, buffalopox, sealpox, canarypox, raccoonpox,pigeonpox and rabbitpox. Poxvirus further include live and attenuatedpathogenic or non-pathogenic Vaccinia virus. Exemplary Vaccinia virusinclude vaccinia Ankara (MVA), vaccinia virus Lister strain, vacciniavirus Copenhagen strain, vaccinia virus Connaught strain, vaccinia virusBrighton strain, vaccinia virus LC16m8 strain, vaccinia virus LC16MO,vaccinia virus IHD-J, vaccinia virus Dairen I, vaccinia virus NYCBOHstrain, vaccinia virus Wyeth strain, vaccinia virus Tian Tan, vacciniavirus LIVP, vaccinia virus L-IPV, vaccinia virus Dryvax® and ACAM1000.

Methods of the invention include methods of treatment that results in abeneficial effect. Particular non-limiting examples of beneficialeffects include providing a subject with partial or complete protectionagainst a poxvirus infection or pathogenesis, or a symptom caused by apoxvirus infection or pathogenesis (e.g., inhibit or reduce probabilityor susceptibility). Particular non-limiting examples of beneficialeffects also include reducing, decreasing, inhibiting, delaying orpreventing poxvirus infection or pathogenesis, and reducing, decreasing,inhibiting, ameliorating or preventing onset, severity, duration,progression, frequency or probability of one or more symptoms orpathologies associated with a poxvirus infection or pathogenesis.Additional non-limiting examples of beneficial effects also includereducing, decreasing, amounts of, or inhibiting, delaying or preventingincreases in poxvirus titer or load, proliferation or replication.Further non-limiting particular examples of beneficial effects includereducing, decreasing, inhibiting, delaying, ameliorating or preventingonset, progression, severity, duration, frequency, probability orsusceptibility of a subject to poxvirus infection or pathogenesis, oraccelerating, facilitating or hastening recovery of a subject frompoxvirus infection or pathogenesis or one or more associated symptoms orpathologies.

Methods of the invention therefore include providing a beneficial ortherapeutic effect to a subject, for example, reducing, decreasing,inhibiting, delaying, ameliorating or preventing onset, progression,severity, duration, frequency or probability of poxvirus infection orpathogenesis or one or more symptoms or pathologies associated with orcaused by poxvirus infection or pathogenesis; reducing, decreasing,inhibiting, delaying or preventing increases in poxvirus titer, load,replication, proliferation, or an amount of a viral protein of one ormore poxvirus strains or isolates or subtypes. Stabilizing theinfection, a symptom or pathology thereof, or preventing, inhibiting ordelaying a worsening or progression of the infection or a symptom orpathology associated with or caused by poxvirus infection orpathogenesis, or progression of the underlying poxvirus infection, arealso included in various embodiments of the methods of the invention.

Specific examples of symptoms and pathologies associated with or causedby poxvirus infection or pathogenesis, whose onset, progression,severity, frequency, duration or probability can be reduced, decreasedinhibited, delayed ameliorated or prevented include, for example, highfever, muscle aches, fatigue, headache, backache, malaise, rash(maculopapular, vesicular or pustular), blisters, pustules or lesions,delirium, vomiting, diarrhea and excess bleeding. Other symptoms andpathologies of poxvirus infection or pathogenesis, are known in the artand treatment thereof in accordance with the invention is provided.

The methods of the invention, including, among other methods, providinga subject with protection against a poxvirus infection or pathogenesis,treatment of a poxvirus infection or pathogenesis, or a symptom orpathology associated with or caused by poxvirus infection orpathogenesis, or decreasing susceptibility of a subject to a poxvirusinfection or pathogenesis, can therefore result in an improvement in thesubjects' condition. An improvement is therefore any objective orsubjective reduction, decrease, inhibition, delay, ameliorating orprevention of onset, progression, severity, duration, frequency orprobability of one or more symptoms or pathologies associated with orcaused by poxvirus infection or pathogenesis, or virus titer, load,replication, proliferation, or an amount of a viral protein. Animprovement would also include reducing, inhibiting or preventingincreases in virus titer, load, replication, proliferation, or an amountof a viral protein of one or more poxvirus strains or isolates orsubtypes or species. An improvement would further include stabilizing asymptom or pathology associated with or caused by poxvirus infection orpathogenesis, or inhibiting, decreasing, delaying or preventing aworsening or progression of the symptom or pathology associated with orcaused by poxvirus infection or pathogenesis, or progression of theunderlying poxvirus infection. An improvement can therefore be, forexample, in any of high fever, muscle aches, fatigue, headache,backache, malaise, rash, blisters, pustules, lesions, delirium,vomiting, diarrhea, excess bleeding and death to any degree or for anyduration of time (hours, days, weeks, months, years, or cure).

An improvement would also include reducing or eliminating a need, dosageamount or frequency of another treatment, such as an antiviral drug orother agent used for treating a subject having or at risk of having apoxvirus infection or pathogenesis, a symptom or pathology associatedwith or caused by poxvirus infection or pathogenesis, or decreasing orpreventing an adverse side effect caused by vaccination with or againsta poxvirus. Thus, reducing an amount of another treatment for poxvirusinfection or pathogenesis, a symptom or pathology associated with orcaused by poxvirus, or an adverse side effect caused by vaccination withor against a poxvirus is considered to provide a benefit and, therefore,is considered within the invention methods. Non-limiting exemplarypoxvirus treatments that may be eliminated or used at reduced doses orfrequencies of administration include vaccinia immune globulin (VIG),cidofivir, or an antibody that binds to a poxvirus protein (e.g., avaccinia virus protein). Additional non-limiting exemplary poxvirustreatments include vaccination, such as with an attenuated or livepoxvirus, for example, modified vaccinia Ankara (MVA), vaccinia virusLister strain, vaccinia virus LC16m8 strain, vaccinia virus NYCBOHstrain, vaccinia virus Wyeth strain or vaccinia virus Dryvax®, or a,vaccinia virus protein used for immunization of a subject againstpoxvirus infection or pathogenesis.

A treatment or improvement need not be complete ablation of anyparticular infection, pathogenesis, symptom, pathology or adverse sideeffect, or all of the infection, pathology, symptoms, pathologies oradverse side effects associated with or caused by poxvirus infection orpathogenesis, or vaccination against a poxvirus. Rather, treatment maybe any objective or subjective measurable or detectable anti-viruseffect or improvement in a treated subject. Thus, reducing, inhibitingdecreasing, eliminating, delaying, halting or preventing a progressionor worsening of the infection or pathogenesis, a symptom or pathology ofthe infection or pathogenesis, or an adverse side effect caused byvaccination is a satisfactory outcome. For example, a compound of theinvention (e.g., CSA) may reduce, delay or stabilize high fever, but nothave any effect on fatigue, muscle aches, fatigue, headache, backache,malaise, rash, blisters, pustules, lesions, delirium, vomiting, diarrheaor excess bleeding. Another example is where a compound of the inventionreduces fatigue, without a detectable improvement in or more othersymptoms or pathologies. Thus, a satisfactory clinical endpoint isachieved when there is an incremental improvement in the subject'scondition or a partial reduction or a stabilization of a poxvirusinfection, pathogenesis or a symptom, pathology or adverse side effectthereof, or an inhibition or prevention of worsening or progression ofthe poxvirus infection, pathogenesis, symptom, pathology or adverse sideeffect thereof (stabilizing one or more symptoms or pathologies), over ashort or long duration (hours, days, weeks, months, years, or cure).

In the methods of the invention in which there is a desired outcome, forexample, a therapeutic or prophylactic method that provides an objectiveor subjective improvement in a poxvirus infection or pathogenesis, asymptom or pathology associated with or caused by poxvirus, or anadverse side effect caused by vaccination with or against a poxvirus, acompound of the invention (e.g., CSA) can be administered in asufficient or effective amount. As used herein, a “sufficient amount” or“effective amount” or an “amount sufficient” or an “amount effective”refers to an amount that provides, in single or multiple doses, alone orin combination with one or more other compounds, treatments, agents(e.g., a drug) or therapeutic regimens, a long term or a short termdetectable or measurable improvement or beneficial effect to a givensubject of any degree or for any time period or duration (e.g., forminutes, hours, days, months, years, or cured).

A “sufficient amount” or “effective amount” therefore includesdecreasing, reducing, inhibiting, preventing, or delaying onset;decreasing, reducing, inhibiting, delaying, or preventing a progressionor worsening of; or reducing, relieving, ameliorating, or alleviating,severity, frequency, duration, susceptibility or probability of poxvirusinfection or pathogenesis, one or more symptoms associated with orcaused by poxvirus infection or pathogenesis, or an adverse side effectof vaccination with or against a poxvirus. In addition, hastening asubject's recovery poxvirus infection or pathogenesis, one or moresymptoms associated with or caused by poxvirus infection orpathogenesis, or an adverse side effect of vaccination with or against apoxvirus is considered to be a sufficient or effective amount. Variousbeneficial effects and indicia of therapeutic and prophylactic benefitare as set forth herein and are known to the skilled artisan.

A sufficient amount or an effective amount can but need not be providedin a single administration and can but need not be administered alone(i.e., without a second drug, agent, treatment or therapeutic regimen),or in combination with another compound, agent, treatment or therapeuticregimen. In addition, a sufficient amount or an effective amount neednot be sufficient or effective if given in single or multiple doseswithout a second compound, treatment, agent, or therapeutic regimen,since additional doses, amounts, frequency or duration of administrationabove and beyond such doses, or additional compounds, agents, treatmentsor therapeutic regimens may be included in order to be effective orsufficient in a given subject.

A sufficient amount or an effective amount need not be effective in eachand every subject, nor a majority of subjects in a given group orpopulation. Thus, a sufficient amount or an effective amount meanssufficiency or effectiveness in a particular subject, not a group or thegeneral population. As is typical for such methods, some subjects willexhibit a greater or less response to a method of the invention thanother subjects.

Amounts, frequencies or duration also considered sufficient andeffective and are therefore beneficial are those that result in theelimination or a reduction in amount, frequency or duration of anothercompound, agent, treatment or therapeutic regimen. For example, acompound of the invention is considered as having a beneficial ortherapeutic effect if contact, administration or delivery in vivoresults in the use of a lesser amount, frequency or duration of anothercompound, agent, treatment or therapeutic regimen to treat theinfection, pathogenesis, symptom or pathology, or adverse side effect ofvaccination.

Any compound, agent, treatment or other therapeutic regimen having abeneficial, additive, synergistic or complementary activity or effectcan be formulated or used in combination with or in addition to theinvention compounds (e.g., CSAs). In various embodiments, the compound,agent, treatment or therapeutic regimen is for providing a subject withprotection against a poxvirus infection or pathogenesis; treating asubject for poxvirus infection or pathogenesis; decreasingsusceptibility of a subject to a poxvirus infection or pathogenesis; ordecreasing or preventing an adverse side effect caused by poxvirusvaccination. Thus, compositions of the invention include CSAcombinations with other CSAs, CSA combinations with other agents ortreatments (e.g., anti-poxvirus drugs, such as cidofivir, poxvirusvaccines, poxvirus proteins, poxvirus antibodies, VIG, etc.), andmethods of the invention include contact with, administration in vitroor in vivo, with another compound (e.g., another CSA), agent, treatmentor therapeutic regimen appropriate for the condition to be treated. Thecompound (e.g., another CSA), agent, treatment or therapeutic regimenappropriate may be used in accordance with the prophylactic andtherapeutic treatment methods, as well as methods for decreasing orpreventing an adverse side effect caused by poxvirus vaccination, as setforth herein, prior to, concurrently or following contacting oradministering a compound of the invention (e.g., CSA) in vitro or invivo.

Examples of such combination compositions and methods include VIG andcidofivir. Additional non-limiting combination compositions and methodsinclude poxvirus vaccination, such as with an attenuated or livepoxvirus, for example, modified vaccinia Ankara (MVA), vaccinia virusLister strain, vaccinia virus LC16m8 strain, vaccinia virus NYCBOHstrain, vaccinia virus Wyeth strain or vaccinia virus Dryvax®, or avaccinia virus protein used for immunization of a subject againstpoxvirus infection or pathogenesis. Further combination compositions andmethods include poxvirus protein or antibody that binds to a poxvirusproteins (e.g., a vaccinia virus protein). A pool of poxvirus proteinsor poxvirus binding antibodies (e.g., monoclonal or polyclonal) can becombined with a compound of the invention or administered separately(prior to, concurrently with or following) administration of a compoundin accordance with the invention. In particular embodiments, anadditional poxvirus protein is present on one or more of intracellularmature virion (IMV), cell-associated enveloped virion (CEV) orextracellular enveloped virion (EEV) forms of smallpox. In additionalparticular aspects, an additional poxvirus protein is one or more ofvaccinia B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, A28, A14, A56,A34, A36, A2, or a B5R, L1R, D8L, A27L, A17L, L5, A21, H2, A28, A14,A56, A34, A36, or A2 homolog. In further particular embodiments, anadditional antibody binds to a poxvirus protein present on one or moreof intracellular mature virion (IMV), cell-associated enveloped virion(CEV) or extracellular enveloped virion (EEV) forms of smallpox. Instill further particular embodiments, an additional antibody binds to apoxvirus protein, such as vaccinia protein B5R, L1R, D8L, A33R, A27L,A17L, L5, A21, H2, A28, A14, A56, A34, A36, A2, or a B5R, L1R, D8L,A33R, A27L, A17L, L5, A21, H2, A28, A14, A56, A34, A36, or A2 homolog.

Antibodies include proteins that bind to other molecules (antigens) viaheavy and light chain variable domains, V_(H) and V_(L), respectively.An antibody is any polyclonal or monoclonal immunoglobulin molecule, ormixture thereof, such as IgM, IgG, IgA, IgE, IgD, and any subclassthereof, such as IgG₁, IgG₂, IgG₃, IgG₄, etc. A monoclonal antibody,refers to an antibody that is based upon, obtained from or derived froma single clone, including any eukaryotic, prokaryotic, or phage clone.An antibody also includes a functional (e.g., binding) fragment orsubsequence, such as, for example, Fab, Fab′, F(ab′)₂, Fv, Fd, scFv andsdFv, unless otherwise expressly stated.

Antibodies include those specific or selective for binding to poxvirusprotein or a homolog. That is, binding to proteins other than thepoxvirus protein or a homolog is such that the binding does notsignificantly interfere with detection of the poxvirus protein orhomolog, unless such other proteins have a similar or same epitope thepoxvirus protein or homolog that is recognized by the poxvirus antibody.Selective binding can be distinguished from non-selective binding usingspecificity, affinity and other binding assays, competitive andnon-competitive, known in the art.

Antibodies include “human” forms, which mean that the amino acidsequence of the antibody is fully human or can or do exist in a humanantibody. An antibody that is non-human may be made fully human bysubstituting non-human amino acid residues with amino acid residues thatcan or do exist in a human antibody. Amino acid residues present inhuman antibodies, CDR region maps and human antibody consensus residuesare known in the art (see, e.g., Kabat, Sequences of Proteins ofImmunological Interest, 4^(th) Ed. US Department of Health and HumanServices. Public Health Service (1987); Chothia and Lesk J. Mol. Biol.186:651 (1987); Padlan Mol. Immunol. 31:169 (1994); and Padlan Mol.Immunol. 28:489 (1991)).

Antibodies include “human” forms, which means that the amino acidsequence of the antibody has non-human amino acid residues (e.g., mouse,rat, goat, rabbit, etc.) of one or more complementarity determiningregions (CDRs) that specifically bind to the desired antigen in anacceptor human immunoglobulin molecule, and one or more human amino acidresidues in the Fv framework region (FR), which are amino acid residuesthat flank the CDRs. Antibodies referred to as “primatized” in the artare within the meaning of “humanized” as used herein, except that theacceptor human immunoglobulin molecule and framework region amino acidresidues may be any primate amino acid residue (e.g., ape, gibbon,gorilla, chimpanzees orangutan, macaque), in addition to any humanresidue.

Antibodies include “chimeric” forms, which means that the amino acidsequence of the antibody contains one or more portions that are derivedfrom, obtained or isolated from, or based upon two or more differentspecies. That is, for example, a portion of the antibody may be human(e.g., a constant region) and another portion of the antibody may benon-human (e.g., a murine heavy or light chain variable region). Thus, achimeric antibody is a molecule in which different portions of theantibody are of different species origins. Unlike a humanized antibody,a chimeric antibody can have the different species sequences in anyregion of the antibody.

The term “subject” refers to an animal, typically mammalian animals,such as but not limited to non-human primates (apes, gibbons, gorillas,chimpanzees, orangutans, macaques), domestic animals (dogs and cats), afarm animals (chickens, ducks, horses, cows, goats, sheep, pigs),experimental animal (mouse, rat, rabbit, guinea pig) and humans.Subjects include animal models, for example, a mouse model of poxvirusinfection (e.g., vaccinia virus). Subjects include naturally occurringor non-naturally occurring mutated or non-human genetically engineered(e.g., transgenic or knockout) animals. Subjects further include animalshaving or at risk of having a chronic or acute poxvirus infection orpathogenesis, symptom of poxvirus infection or pathogenesis, or adverseside effect caused by vaccination with or against poxvirus. Subjects canbe any age. For example, a subject (e.g., human) can be a newborn,infant, toddler, child, teenager, or adult, e.g., 50 years or older.

Subjects include those in need of a method of the invention, e.g., inneed of a therapeutic or prophylactic treatment. A subject is consideredto be in need of a method of the invention where a method is likely toprovide some benefit to a subject. Various benefits provided to asubject are as set forth herein and known in the art for poxvirusinfection, pathogenesis, symptoms or pathologies caused by or associatedwith poxvirus infection or pathogenesis, and adverse side effects causedby vaccination with or against a poxvirus.

Subjects appropriate for treatment include those having poxvirusinfection or pathogenesis or having any symptom or pathology associatedwith or caused by poxvirus. Target subjects therefore include subjectsthat have been infected with poxvirus, or that have developed one ormore adverse symptoms or pathologies associated with or caused bypoxvirus infection or pathogenesis, regardless of the virus type, timingor degree of onset, progression, severity, frequency, duration of anyinfection, pathogenesis, symptom, pathology or adverse side effect.

Subjects appropriate for treatment also include those at risk ofpoxvirus infection or pathogenesis or at risk of having or developing apoxvirus infection. Candidate subjects therefore include subjects thathave been exposed to or contacted with poxvirus, or that are at risk ofexposure to or contact with poxvirus, regardless of the type, timing orextent of exposure or contact. The invention methods are thereforeapplicable to a subject who is at risk of poxvirus infection orpathogenesis, but has not yet been exposed to or contacted withpoxvirus. Prophylactic methods are therefore included. Subjects targetedfor prophylaxis can be at increased risk (probability or susceptibility)of poxvirus infection or pathogenesis, as set forth herein and known inthe art.

At risk subjects appropriate for treatment include subjects exposed toother subjects having a poxvirus, or where the risk of poxvirusinfection is increased due to changes in virus infectivity or celltropism, immunological susceptibility (e.g., an immunocompromisedsubject), or environmental risk. At risk subjects appropriate fortreatment therefore include human subjects exposed to or at risk ofexposure to other humans that have a poxvirus infection, or are at riskof a poxvirus infection in the environment.

Subjects also appropriate for treatment also include those vaccinatedagainst or a candidate for vaccination against poxvirus (e.g.,vaccinated with live or attenuated Vaccinia virus). Subjects thereforeinclude vaccinated subjects that have not or have been exposed to orcontacted with poxvirus, as well as candidate subjects for vaccinationthat have not or have been exposed to or contacted with poxvirus,regardless of the type, timing or extent of exposure or contact.

In various embodiments, a subject has or is a candidate for vaccinationwith a live or attenuated Vaccinia virus. In various aspects, thesubject is a candidate for or has been vaccinated with a modifiedvaccinia Ankara (MVA), vaccinia virus Lister strain, vaccinia virusLC16m8 strain, vaccinia virus NYCBOH strain, vaccinia virus Wyethstrain, vaccinia virus Dryvax® or ACAM1000. In further aspects, asubject is administered a compound of the invention (e.g., CSA) priorto, concurrently with, or following vaccination (e.g., within 0-2, 2-4,4-12 or 12-24 hours or days of vaccination).

Subjects further include immunocompromised subjects due to animmunological disorder (e.g., autoimmunity) or disease, or animmune-suppressing treatment (e.g., cyclophosphamide). Subjects alsoinclude those having been exposed to or diagnosed as HIV+. Subjectsfurther include those receiving or candidates for a tissue or organtransplant.

Compounds of the invention, including CSAs, can be incorporated intopharmaceutical compositions or formulations. Such pharmaceuticalcompositions/formulations are useful for administration to a subject, invivo or ex vivo.

Pharmaceutical compositions and formulations include carriers orexcipients for administration to a subject. As used herein the terms“pharmaceutically acceptable” and “physiologically acceptable” mean abiologically compatible formulation, gaseous, liquid or solid, ormixture thereof, which is suitable for one or more routes ofadministration, in vivo delivery or contact. A formulation is compatiblein that it does not destroy activity of an active ingredient therein(e.g., a CSA), or induce adverse side effects that far outweigh anyprophylactic or therapeutic effect or benefit.

Such formulations include solvents (aqueous or non-aqueous), solutions(aqueous or non-aqueous), emulsions (e.g., oil-in-water orwater-in-oil), suspensions, syrups, elixirs, dispersion and suspensionmedia, coatings, isotonic and absorption promoting or delaying agents,compatible with pharmaceutical administration or in vivo contact ordelivery. Aqueous and non-aqueous solvents, solutions and suspensionsmay include suspending agents and thickening agents. Suchpharmaceutically acceptable carriers include tablets (coated oruncoated), capsules (hard or soft), microbeads, powder, granules andcrystals. Supplementary active compounds (e.g., preservatives,antibacterial, antiviral and antifungal agents) can also be incorporatedinto the compositions.

The formulations may, for convenience, be prepared or provided as a unitdosage form. Preparation techniques include bringing into associationthe active ingredient (e.g., CSA) and a pharmaceutical carrier(s) orexcipient(s). In general, formulations are prepared by uniformly andintimately associating the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product. For example, a tablet may be made by compression ormolding. Compressed tablets may be prepared by compressing, in asuitable machine, an active ingredient (e.g., a CSA) in a free-flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Molded tablets may be produced by molding, in a suitableapparatus, a mixture of powdered compound (e.g., CSA) moistened with aninert liquid diluent. The tablets may optionally be coated or scored andmay be formulated so as to provide a slow or controlled release of theactive ingredient therein.

Cosolvents and adjuvants may be added to the formulation. Non-limitingexamples of cosolvents contain hydroxyl groups or other polar groups,for example, alcohols, such as isopropyl alcohol; glycols, such aspropylene glycol, polyethyleneglycol, polypropylene glycol, glycolether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acidesters. Adjuvants include, for example, surfactants such as, soyalecithin and oleic acid; sorbitan esters such as sorbitan trioleate; andpolyvinylpyrrolidone.

Supplementary active compounds (e.g., preservatives, antioxidants,antimicrobial agents including biocides and biostats such asantibacterial, antiviral and antifungal agents) can also be incorporatedinto the compositions. Preservatives and other additives include, forexample, antimicrobials, anti-oxidants, chelating agents and inert gases(e.g., nitrogen). Pharmaceutical compositions may therefore includepreservatives, antimicrobial agents, anti-oxidants, chelating agents andinert gases.

Preservatives can be used to inhibit microbial growth or increasestability of the active ingredient thereby prolonging the shelf life ofthe pharmaceutical formulation. Suitable preservatives are known in theart and include, for example, EDTA, EGTA, benzalkonium chloride orbenzoic acid or benzoates, such as sodium benzoate. Antioxidantsinclude, for example, ascorbic acid, vitamin A, vitamin E, tocopherols,and similar vitamins or provitamins.

An antimicrobial agent or compound directly or indirectly inhibits,reduces, delays, halts, eliminates, arrests, suppresses or preventscontamination by or growth, infectivity, replication, proliferation,reproduction, of a pathogenic or non-pathogenic microbial organism.Classes of antimicrobials include, antibacterial, antiviral, antifungaland antiparasitics. Antimicrobials include agents and compounds thatkill or destroy (-cidal) or inhibit (-static) contamination by orgrowth, infectivity, replication, proliferation, reproduction of themicrobial organism.

Exemplary antibacterials (antibiotics) include penicillins (e.g.,penicillin G, ampicillin, methicillin, oxacillin, and amoxicillin),cephalosporins (e.g., cefadroxil, ceforanid, cefotaxime, andceftriaxone), tetracyclines (e.g., doxycycline, chlortetracycline,minocycline, and tetracycline), aminoglycosides (e.g., amikacin,gentamycin, kanamycin, neomycin, streptomycin, netilmicin, paromomycinand tobramycin), macrolides (e.g., azithromycin, clarithromycin, anderythromycin), fluoroquinolones (e.g., ciprofloxacin, lomefloxacin, andnorfloxacin), and other antibiotics including chloramphenicol,clindamycin, cycloserine, isoniazid, rifampin, vancomycin, aztreonam,clavulanic acid, imipenem, polymyxin, bacitracin, amphotericin andnystatin.

Particular non-limiting classes of anti-virals include reversetranscriptase inhibitors; protease inhibitors; thymidine kinaseinhibitors; sugar or glycoprotein synthesis inhibitors; structuralprotein synthesis inhibitors; nucleoside analogues; and viral maturationinhibitors. Specific non-limiting examples of anti-virals include thoseset forth above and, nevirapine, delavirdine, efavirenz, saquinavir,ritonavir, indinavir, nelfinavir, amprenavir, zidovudine (AZT),stavudine (d4T), larnivudine (3TC), didanosine (DDI), zalcitabine (ddC),abacavir, acyclovir, penciclovir, valacyclovir, ganciclovir, 1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9->2-hydroxy-ethoxymethylguanine, adamantanamine, 5-iodo-2′-deoxyuridine,trifluorothymidine, interferon and adenine arabinoside.

Exemplary antifungals include agents such as benzoic acid, undecylenicalkanolamide, ciclopiroxolamine, polyenes, imidazoles, allylamine,thicarbamates, amphotericin B, butylparaben, clindamycin, econaxole,amrolfine, butenafine, naftifine, terbinafine, ketoconazole, elubiol,econazole, econaxole, itraconazole, isoconazole, miconazole,sulconazole, clotrimazole, enilconazole, oxiconazole, tioconazole,terconazole, butoconazole, thiabendazole, voriconazole, saperconazole,sertaconazole, fenticonazole, posaconazole, bifonazole, fluconazole,flutrimazole, nystatin, pimaricin, amphotericin B, flucytosine,natamycin, tolnaftate, mafenide, dapsone, caspofungin, actofunicone,griseofulvin, potassium iodide, Gentian Violet, ciclopirox, ciclopiroxolamine, haloprogin, ketoconazole, undecylenate, silver sulfadiazine,undecylenic acid, undecylenic alkanolamide and Carbol-Fuchsin.

Pharmaceutical compositions can optionally be formulated to becompatible with a particular route of administration. Thus,pharmaceutical compositions include carriers (excipients, diluents,vehicles or filling agents) suitable for administration by variousroutes and delivery, locally, regionally or systemically.

Exemplary routes of administration for contact or in vivo delivery whicha compound of the invention (e.g., CSA) can optionally be formulatedinclude inhalation, respiration, intubation, intrapulmonaryinstillation, oral (buccal, sublingual, mucosal), intrapulmonary,rectal, vaginal, intrauterine, intradermal, topical, dermal, parenteral(e.g., subcutaneous, intramuscular, intravenous, intradermal,intraocular, intratracheal and epidural), intranasal, intrathecal,intraarticular, intracavity, transdermal, iontophoretic, ophthalmic,optical (e.g., corneal), intraglandular, intraorgan, intralymphatic.

Formulations suitable for parenteral administration include aqueous andnon-aqueous solutions, suspensions or emulsions of the compound, whichmay include suspending agents and thickening agents, which preparationsare typically sterile and can be isotonic with the blood of the intendedrecipient. Non-limiting illustrative examples of aqueous carriersinclude water, saline (sodium chloride solution), dextrose (e.g.,Ringer's dextrose), lactated Ringer's, fructose, ethanol, animal,vegetable or synthetic oils. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers (suchas those based on Ringer's dextrose). The formulations may be presentedin unit-dose or multi-dose kits, for example, ampules and vials, and maybe stored in a freeze-dried (lyophilized) condition requiring additionof a sterile liquid carrier, for example, water for injections, prior touse.

For transmucosal or transdermal administration (e.g., topical contact),penetrants can be included in the pharmaceutical composition. Penetrantsare known in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.For transdermal administration, the active ingredient can be formulatedinto aerosols, sprays, ointments, salves, gels, pastes, lotions, oils orcreams as generally known in the art.

For topical administration, for example, to skin, pharmaceuticalcompositions typically include ointments, creams, lotions, pastes, gels,sprays, aerosols or oils. Carriers which may be used include Vaseline,lanolin, polyethylene glycols, alcohols, transdermal enhancers, andcombinations thereof. An exemplary topical delivery system is atransdermal patch containing an active ingredient (e.g., CSA).

For oral administration, pharmaceutical compositions include capsules,cachets, lozenges, tablets or troches, as powder or granules. Oraladministration formulations also include a solution or a suspension(e.g., aqueous liquid or a non-aqueous liquid; or as an oil-in-waterliquid emulsion or a water-in-oil emulsion).

For airway or nasal administration, pharmaceutical compositions can beformulated in a dry powder for delivery, such as a fine or a coarsepowder having a particle size, for example, in the range of 20 to 500microns which is administered in the manner by inhalation through theairways or nasal passage. Depending on delivery device efficiency,effective dry powder dosage levels typically fall in the range of about10 to about 100 mg. Appropriate formulations, wherein the carrier is aliquid, for administration, as for example, a nasal spray or as nasaldrops, include aqueous or oily solutions of the active ingredient.

For airway or nasal administration, aerosol and spray delivery systemsand devices, also referred to as “aerosol generators” and “spraygenerators,” such as metered dose inhalers (MDI), nebulizers(ultrasonic, electronic and other nebulizers), nasal sprayers and drypowder inhalers can be used. MDIs typically include an actuator, ametering valve, and a container that holds a suspension or solution,propellant, and surfactant (e.g., oleic acid, sorbitan trioleate,lecithin). Activation of the actuator causes a predetermined amount tobe dispensed from the container in the form of an aerosol, which isinhaled by the subject. MDIs typically use liquid propellant andtypically, MDIs create droplets that are 15 to 30 microns in diameter,optimized to deliver doses of 1 microgram to 10 mg of a therapeutic.Nebulizers are devices that turn medication into a fine mist inhalableby a subject through a face mask that covers the mouth and nose.Nebulizers provide small droplets and high mass output for delivery toupper and lower respiratory airways. Typically, nebulizers createdroplets down to about 1 micron in diameter.

Dry-powder inhalers (DPI) can be used to deliver the compounds of theinvention, either alone or in combination with a pharmaceuticallyacceptable carrier. DPIs deliver active ingredient to airways and lungswhile the subject inhales through the device. DPIs typically do notcontain propellants or other ingredients, only medication, but mayoptionally include other components. DPIs are typicallybreath-activated, but may involve air or gas pressure to assistdelivery.

For rectal administration, pharmaceutical compositions can be includedas a suppository with a suitable base comprising, for example, cocoabutter or a salicylate. For vaginal administration, pharmaceuticalcompositions can be included as pessaries, tampons, creams, gels,pastes, foams or spray formulations containing in addition to the activeingredient (e.g., CSA) a carrier, examples of appropriate carriers whichare known in the art.

Pharmaceutical formulations and delivery systems appropriate for thecompositions and methods of the invention are known in the art (see,e.g., Remington: The Science and Practice of Pharmacy (2003) 20^(th)ed., Mack Publishing Co., Easton, Pa.; Remington's PharmaceuticalSciences (1990) 18^(th) ed., Mack Publishing Co., Easton, Pa.; The MerckIndex (1996) 12^(th) ed., Merck Publishing Group, Whitehouse, N.J.;Pharmaceutical Principles of Solid Dosage Forms (1993), TechnonicPublishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, PharmaceuticalCalculations (2001) 11^(th) ed., Lippincott Williams & Wilkins,Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, N.Y., pp. 253-315).

Compounds of the invention (e.g., CSAs), including pharmaceuticalformulations can be packaged in unit dosage forms for ease ofadministration and uniformity of dosage. A “unit dosage form” as usedherein refers to a physically discrete unit suited as unitary dosagesfor the subject to be treated; each unit containing a predeterminedquantity of compound optionally in association with a pharmaceuticalcarrier (excipient, diluent, vehicle or filling agent) which, whenadministered in one or more doses, is calculated to produce a desiredeffect (e.g., prophylactic or therapeutic effect or benefit). Unitdosage forms can contain a daily dose or unit, daily sub-dose, or anappropriate fraction thereof, of an administered compound (e.g., CSA).Unit dosage forms also include, for example, capsules, troches, cachets,lozenges, tablets, ampules and vials, which may include a composition ina freeze-dried or lyophilized state; a sterile liquid carrier, forexample, can be added prior to administration or delivery in vivo. Unitdosage forms additionally include, for example, ampules and vials withliquid compositions disposed therein. Unit dosage forms further includecompounds for transdermal administration, such as “patches” that contactwith the epidermis of the subject for an extended or brief period oftime. The individual unit dosage forms can be included in multi-dosekits or containers. Pharmaceutical formulations can be packaged insingle or multiple unit dosage forms for ease of administration anduniformity of dosage.

Compounds of the invention (e.g., CSAs) can be administered inaccordance with the methods at any frequency as a single bolus ormultiple dose e.g., one, two, three, four, five, or more times hourly,daily, weekly, monthly or annually or between about 1 to 10 days, weeks,months, or for as long as appropriate. Exemplary frequencies aretypically from 1-7 times, 1-5 times, 1-3 times, 2-times or once, daily,weekly or monthly. Timing of contact, administration ex vivo or in vivodelivery can be dictated by the infection, pathogenesis, symptom,pathology or adverse side effect to be treated. For example, an amountcan be administered to the subject substantially contemporaneously with,or within about 1-60 minutes or hours of the onset of a symptom oradverse side effect of poxvirus infection, pathogenesis or vaccination.

Doses may vary depending upon whether the treatment is therapeutic orprophylactic, the onset, progression, severity, frequency, duration,probability of or susceptibility of the symptom, the type of virusinfection or pathogenesis to which treatment is directed, clinicalendpoint desired, previous, simultaneous or subsequent treatments,general health, age, gender or race of the subject, bioavailability,potential adverse systemic, regional or local side effects, the presenceof other disorders or diseases in the subject, and other factors thatwill be appreciated by the skilled artisan (e.g., medical or familialhistory). Dose amount, frequency or duration may be increased orreduced, as indicated by the clinical outcome desired, status of theinfection, symptom or pathology, any adverse side effects of thetreatment or therapy. The skilled artisan will appreciate the factorsthat may influence the dosage, frequency and timing required to providean amount sufficient or effective for providing a prophylactic ortherapeutic effect or benefit.

Typically, for therapeutic treatment, a compound of the invention (e.g.,CSA) will be administered as soon as practical, typically within 0-72hours after a subject is exposed to or contacted with any poxvirus, orwithin 0-72 hours after development of one or more symptoms orpathologies associated with poxvirus infection or pathogenesis (e.g.,onset of fever, rash, lesions, etc.) or a symptom associated withpathogenic poxviruses such as smallpox and monkeypox.

For prophylactic treatment, a compound of the invention can beadministered immediately or within 0-72 after suspected contact with, or0-4 weeks, e.g., 1-3 weeks, prior to anticipated or possible exposure toor contact with poxvirus. For prophylactic treatment in connection withimmunization/vaccination of a subject, a compound can be administeredprior to, concurrently with or following immunization/vaccination of thesubject.

Doses can be based upon current existing active or passive immunizationprotocols (e.g., VIG), empirically determined, determined using animaldisease models or optionally in human clinical studies. For example,initial study doses can be based upon animal studies, such as a mouse,which weighs about 30 grams, and the amount of compound administered toachieve a prophylactic or therapeutic effect or benefit. The dose can beadjusted according to the mass of a subject, and will generally be in arange from about 0.1-1 ug/kg, 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg,50-100 ug/kg, 100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg,10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, ormore, of subject body weight, two, three, four, or more times per hour,day, week, month or annually. Of course, doses can be more or less, asappropriate, for example, 0.00001 mg/kg of subject body weight to about10,000.0 mg/kg of subject body weight, about 0.001 mg/kg, to about 100mg/kg, about 0.01 mg/kg, to about 10 mg/kg, or about 0.1 mg/kg, to about1 mg/kg of subject body weight over a given time period, e.g., 1, 2, 3,4, 5 or more hours, days, weeks, months, years. A subject may beadministered in single bolus or in divided/metered doses, which can beadjusted to be more or less according to the various consideration setforth herein and known in the art.

Dose amount, frequency or duration may be increased or reduced, asindicated by the status of the poxvirus infection or pathogenesis,associated symptom or pathology, or any adverse side effect(s) ofvaccination, treatment or anti-poxvirus therapy. For example, oncecontrol or a particular endpoint is achieved, for example, reducing,decreasing, inhibiting, ameliorating or preventing onset, severity,duration, progression, frequency or probability of one or more symptomsassociated with a poxvirus infection or pathogenesis of one or moresymptoms or pathologies associated with or caused by poxvirus infectionor pathogenesis, dose amount, frequency or duration can be reduced.

The invention provides kits including compounds of the invention (e.g.,CSA), combination compositions and pharmaceuticalcompositions/formulations thereof, packaged into a suitable packagingmaterial. In one embodiment, a kit includes packaging material, acationic steroid antimicrobial (CSA) and instructions. In variousaspects, the instructions are for administering the CSA to: provide asubject with protection against a poxvirus infection or pathogenesis;treat a subject for poxvirus infection or pathogenesis; decreasesusceptibility of a subject to a poxvirus infection or pathogenesis; ordecrease or prevent an adverse side effect caused by vaccination of asubject with a poxvirus.

The term “packaging material” refers to a physical structure housing oneor more components of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,vials, tubes, etc.). A kit can contain a plurality of components, e.g.,two or more compounds of the invention alone or in combination with ananti-poxvirus agent or treatment (e.g., an anti-viral, a poxvirusprotein or an antibody that binds to a poxvirus protein) or drug,optionally sterile.

A kit optionally includes a label or insert including a description ofthe components (type, amounts, doses, etc.), instructions for use invitro, in vivo, or ex vivo, and any other components therein. Labels orinserts include “printed matter,” e.g., paper or cardboard, or separateor affixed to a component, a kit or packing material (e.g., a box), orattached to an ampule, tube or vial containing a kit component. Labelsor inserts can additionally include a computer readable medium, such asa disk (e.g., floppy diskette, hard disk, ZIP disk), optical disk suchas CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storagemedia such as RAM and ROM or hybrids of these such as magnetic/opticalstorage media, FLASH media or memory type cards.

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics andpharmacodynamics. Labels or inserts can include information identifyingmanufacturer, lot numbers, manufacturer location and date, expirationdates.

Labels or inserts can include information on a condition, disorder ordisease (e.g., virus pathogenesis or infection) for which a kitcomponent may be used. Labels or inserts can include instructions for aclinician or subject for using one or more of the kit components in amethod, treatment protocol or therapeutic/prophylactic regimen,including the methods of the invention. Instructions can include amountsof compound, frequency or duration of administration, and instructionsfor practicing any of the methods, treatment protocols or prophylacticor therapeutic regimes described herein. Exemplary instructions include,instructions for treating poxvirus infection or pathogenesis. Kits ofthe invention therefore can additionally include labels or instructionsfor practicing any of the methods of the invention described hereinincluding treatment, screening or other methods. Thus, for example, akit can include a compound of the invention (e.g., CSA) that has one ormore anti-poxvirus activities as set forth herein, together withinstructions for administering the compound in a prophylactic ortherapeutic treatment method of the invention, for example to a subjectin need of such treatment. Exemplary instructions include administeringthe CSA to: provide a subject with protection against a poxvirusinfection or pathogenesis; treat a subject for poxvirus infection orpathogenesis; decrease susceptibility of a subject to a poxvirusinfection or pathogenesis; or decrease or prevent an adverse side effectcaused by vaccination of a subject with a poxvirus.

Labels or inserts can include information on any effect or benefit a kitcomponent may provide, such as a prophylactic or therapeutic effect orbenefit. For example, a label or insert could provide a description ofone or more symptoms which can be improved, i.e., reducing, decreasing,inhibiting, ameliorating or preventing onset, severity, duration,progression, frequency or probability of one or more symptoms orpathologies associated with a poxvirus infection or pathogenesis, or oneor more adverse side effects associated with poxvirus vaccination.Poxvirus symptoms and pathologies are as set forth herein or known inthe art (e.g., high fever, muscle aches, fatigue, headache, backache,malaise, rash, blisters, pustules, lesions, delirium, vomiting,diarrhea, excess bleeding, death, etc.). Adverse side effects associatedwith poxvirus vaccination are as set forth herein or known in the art(e.g., postvaccinial encephalitis, progressive vaccinia-Vaccinianecrosum, eczema vaccinatum, Vaccinia Keratitis, generalized vaccinia,myopericarditis, inoculation of other sites, infection of closecontacts, rash and periocular infection, death, etc.)

Labels or inserts can include information on potential adverse sideeffects of treatment. Labels or inserts can further include warnings tothe clinician or subject regarding situations or conditions where asubject should stop or reduce use of a particular kit component. Adverseside effects could also occur when the subject has, will be or iscurrently taking one or more other medications that may be incompatiblewith a compound of the invention, or the subject has, will be or iscurrently undergoing another treatment protocol or therapeutic regimenwhich would be incompatible with the compound and, therefore, labels orinserts could include information regarding such side effects orincompatibilities.

Invention kits can additionally include a buffering agent, or apreservative or a stabilizing agent in a pharmaceutical formulationcontaining a compound of the invention. Each component of the kit can beenclosed within an individual container and all of the variouscontainers can be within a single package. Invention kits can bedesigned for cold storage.

Invention kits can include components, such as devices for practicing amethod of the invention or administering a compound of the invention(e.g., CSA) to a subject, ex vivo or in vivo. The device can be adelivery device, such as a syringe, a compressible (e.g., squeezable)tube or dermal patch for mucosal, skin/dermis or corneal delivery, or anaerosol delivery device for administration to lungs or airways.

Compounds useful in accordance with the invention, are described herein,both generically and with particularity, and in U.S. Pat. Nos.6,350,738; 6,486,148; and 6,767,904, which are incorporated herein byreference. Compounds include steroid derivatives, such as cationicsteroid antimicrobials (CSA) that exhibit one or more anti-poxvirusactivities or functions. The skilled artisan will recognize thecompounds within the generic formula set forth herein. Additionalcompounds of the invention having one or more anti-poxvirus activitiesor functions are described and can be characterized using the assays setforth herein and in the art.

Compounds of formula I, also referred to as cationic steroidantimicrobials (CSA), comprise:

wherein:

fused rings A, B, C, and D are independently saturated or fully orpartially unsaturated; and

each of R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, and R₁₇ isindependently selected from the group consisting of hydrogen, hydroxyl,a substituted or unsubstituted (C1-C10) alkyl, (C1-C10) hydroxyalkyl,(C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10) alkylcarboxy-(C1-C10) alkyl,(C1-C10) alkylamino-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10)alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted arylamino-(C1-C10)alkyl, (C1-C10) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, a linkinggroup attached to a second steroid, a substituted or unsubstituted(C1-C10) aminoalkyloxy, a substituted or unsubstituted (C1-C10)aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxamido, H₂N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,(C1-C10) guanidinoalkyl oxy, (C1-C10) quaternaryammoniumalkylcarboxy,and (C1-C10) guanidinoalkyl carboxy, where Q5 is a side chain of anyamino acid (including the side chain of glycine, i.e., H), P.G. is anamino protecting group, and

R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is each independently: deleted when one offused rings A, B, C, or D is unsaturated so as to complete the valencyof the carbon atom at that site, or

selected from the group consisting of hydrogen, hydroxyl, a substitutedor unsubstituted (C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10)alkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)aminoalkyl, a substituted or unsubstituted aryl, C1-C10 haloalkyl, C2-C6alkenyl, C2-C6 alkynyl, oxo, a linking group attached to a secondsteroid, a substituted or unsubstituted (C1-C10) aminoalkyloxy, asubstituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substitutedor unsubstituted (C1-C10) aminoalkylaminocarbonyl, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyloxy, and (C1-C10)guanidinoalkylcarboxy, where Q5 is a side chain of any amino acid, P.G.is an amino protecting group, and

provided that at least two of R₁ through R₁₄ are independently selectedfrom the group consisting of a substituted or unsubstituted (C1-C10)aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted arylamino-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl,(C1-C10) quaternaryammonium alkylcarboxy, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyloxy, and (C1-C10)guanidinoalkylcarboxy; or a pharmaceutically acceptable salt thereof.

A “ring” as used herein can be heterocyclic or carbocyclic. The term“saturated” used herein refers to the fused ring of formula I havingeach atom in the fused ring either hydrogenated or substituted such thatthe valency of each atom is filled. The term “unsaturated” used hereinrefers to the fused ring of formula I where the valency of each atom ofthe fused ring may not be filled with hydrogen or other substituents.For example, adjacent carbon atoms in the fused ring can be doubly boundto each other. Unsaturation can also include deleting at least one ofthe following pairs and completing the valency of the ring carbon atomsat these deleted positions with a double bond; such as R₅ and R₉; R₈ andR₁₀; and R₁₃ and R₁₄.

The term “unsubstituted” used herein refers to a moiety having each atomhydrogenated such that the valency of each atom is filled.

The term “halo” used herein refers to a halogen atom such as fluorine,chlorine, bromine, or iodine.

Examples of amino acid side chains include but are not limited to H(glycine), methyl (alanine), —CH₂—(C═O)—NH₂ (asparagine), —CH₂—SH(cysteine), and —CH(OH)CH₃ (threonine).

An alkyl group is a branched or unbranched hydrocarbon that may besubstituted or unsubstituted. Examples of branched alkyl groups includeisopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl,tert-pentyl, isohexyl. Substituted alkyl groups may have one, two, threeor more substituents, which may be the same or different, each replacinga hydrogen atom. Substituents are halogen (e.g., F, Cl, Br, and I),hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protectedcarboxy, cyano, methylsulfonylamino, alkoxy, acyloxy, nitro, and lowerhaloalkyl.

The term “substituted” used herein refers to moieties having one, two,three or more substituents, which may be the same or different, eachreplacing a hydrogen atom. Examples of substituents include but are notlimited to halogen (e.g., F, Cl, Br, and I), hydroxyl, protectedhydroxyl, amino, protected amino, carboxy, protected carboxy, cyano,methylsulfonylamino, alkoxy, alkyl, aryl, aralkyl, acyloxy, nitro, andlower haloalkyl.

An aryl group is a C6-20 aromatic ring, wherein the ring is made ofcarbon atoms (e.g., C6-C14, C6-10 aryl groups). Examples of haloalkylinclude fluoromethyl, dichloromethyl, trifluoromethyl,1,1-difluoroethyl, and 2,2-dibromoethyl.

An aralkyl group is a group containing 6-20 carbon atoms that has atleast one aryl ring and at least one alkyl or alkylene chain connectedto that ring. An example of an aralkyl group is a benzyl group.

A linking group is any divalent moiety used to link a compound offormula to another steroid, e.g., a second compound of formula I. Anexample of a linking group is (C1-C10) alkyloxy-(C1-C10) alkyl.

Amino-protecting groups are known to those skilled in the art. Ingeneral, the species of protecting group is not critical, provided thatit is stable to the conditions of any subsequent reaction(s) on otherpositions of the compound and can be removed at the appropriate pointwithout adversely affecting the remainder of the molecule. In addition,a protecting group may be substituted for another after substantivesynthetic transformations are complete. Clearly, where a compounddiffers from a compound disclosed herein only in that one or moreprotecting groups of the disclosed compound has been substituted with adifferent protecting group, that compound is within the invention.Further examples and conditions are found in T. W. Greene, ProtectiveGroups in Organic Chemistry, (1st ed., 1981, 2nd ed., 1991).

The invention also includes compounds comprising a ring system of atleast 4 fused rings, where each of the rings has from 5-7 atoms. Thering system has two faces, and contains 3 chains attached to the sameface. Each of the chains contains a nitrogen-containing group that isseparated from the ring system by at least one atom; thenitrogen-containing group is an amino group, e.g., a primary aminogroup, or a guanidino group. The compound can also contain a hydrophobicgroup, such as a substituted (C3-10) aminoalkyl group, a (C1-10)alkyloxy (C3-10) alkyl group, or a (C1-10) alkylamino (C3-10)alkylgroup, attached to the steroid backbone.

For example, the compound may have the formula V, where each of thethree chains 10 containing nitrogen-containing groups is independentlyselected from R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R,₁₅, R₁₆, R₁₇, and R₁₈,defined below.

where:

each of fused rings A, B, C, and D is independently saturated, or isfully or partially unsaturated, provided that at least two of A, B, C,and D are saturated, wherein rings A, B, C, and D form a ring system;

each of m, n, p, and q is independently 0 or 1;

each of R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈ isindependently selected from the group consisting of hydrogen, hydroxyl,a substituted or unsubstituted (C1-C10) alkyl, (C1-C10) hydroxyalkyl,(C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10)alkylcarboxy-(C1-C10 alkyl,(C1-C10) alkylamino-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10)alkylamino, (C1-C10 alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted arylamino-(C1-C10)alkyl, (C1-C10) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, a linkinggroup attached to a second steroid, a substituted or unsubstituted(C1-C10) aminoalkyloxy, a substituted or unsubstituted (C1-C10)aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxamido, H₂N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,(C1-C10) guanidinoalkyl oxy, (C1-C10) quaternaryammoniumalkylcarboxy,and (C1-C10) guanidinoalkyl carboxy, where Q5 is a side chain of anyamino acid (including a side chain of glycine, i.e., H). P.G. is anamino protecting group: and

each of R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is independently: deleted when oneof fused rings A, B, C, or D is unsaturated so as to complete thevalency of the carbon atom at that site, or selected from the groupconsisting of hydrogen, hydroxyl, a substituted or unsubstituted(C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo,a linking group attached to a second steroid, a substituted orunsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted(C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,(C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy, where Q5is a side chain of any amino acid, P.G. is an amino protecting group,

provided that at least three of R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅,R₁₆, R₁₇, and R₁₈ are disposed on the same face of the ring system andare independently selected from the group consisting of a substituted orunsubstituted (C1-C10) aminoalkyl, a substituted or unsubstituted(C1-C10) aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted arylamino-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10)aminoalkylaminocarbonyl, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, a substituted or unsubstituted (C1-C5)aminoalkylcarboxamido, a (C1-C10) quaternaryammoniumalkylcarboxy,H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy,(C1-C10) cyanoalkylox, P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10)guanidinoalkyloxy, and a (C1-C10) guanidinoalkylcarboxy; or apharmaceutically acceptable salt thereof. In various aspects, at leasttwo, or at least, three, of m, n, p, and q are 1.

Compounds set forth herein preserve certain stereochemical andelectronic characteristics found in steroids. The term “sameconfiguration” as used herein refers to substituents on the fusedsteroid having the same stereochemical orientation. For examplesubstituents R₃, R₇ and R₁₂ are all β-substituted or α-substituted.

Compounds of the invention include but are not limited to compoundshaving amine or guanidine groups covalently attached to a steroidbackbone or scaffold at any carbon position, e.g., cholic acid. Invarious embodiments, a group is covalently attached at any one, or more,of positions C3, C7 and C12 of the steroid backbone or scaffold. Inadditional embodiments, a group is absent from any one, or more, ofpositions C3, C7 and C12 of the steroid backbone or scaffold.

Compounds of the invention that include such groups can include atether, the tether having variable chain length or size. As used herein,the terms “tether” or “tethered,” when used in reference to a compoundof the invention, refers to the chain of atoms between the steroidbackbone or scaffold and a terminal amino or guanidine group. In variousembodiments, a tether is covalently attached at any one, or more, ofpositions C3, C7 and C12. In additional embodiments, a tether is lackingat any one, or more, of positions C3, C7 and C12. A tether length mayinclude the heteroatom (O or N) covalently attached to the steroidbackbone.

Other ring systems can also be used, e.g., 5-member fused rings.Compounds with backbones having a combination of 5- and 6-membered ringsare also included in the invention. Amine or guanidine groups can beseparated from the backbone by at least one, two, three, four or moreatoms. The backbone can be used to orient the amine or guanidine groupson one face, or plane, of the steroid. For example, a scheme showing acompound having primary amino groups on one face, or plane, of abackbone is shown below:

Methods of synthesizing compounds of formula I are provided, wherein forexample, at least two of R₁ through R₁₄ are independently selected fromthe group consisting of a substituted or unsubstituted (C1-C10)aminoalkyloxy. In one embodiment, a method includes the step ofcontacting a compound of formula IV,

where at least two of R₁ through R₁₄ are hydroxyl, and the remainingmoieties on the fused rings A, B, C, and D are defined for formula I,with an electrophile to produce an alkyl ether compound of formula IV,wherein at least two of R₁ through R₁₄ are (C1-C10)alkyloxy. The alkylether compounds are converted into an amino precursor compound whereinat least two of R₁ through R₁₄ are independently selected from the groupconsisting of (C1-C10) azidoalkyloxy and (C1-C10) cyanoalkyloxy and theamino precursor compound is reduced to form a compound of formula I.

The electrophiles used in a method include but are not limited to2-(2-bromoethyl)-1,3-dioxolane, 2-iodoacetamide, 2-chloroacetamide,N-(2-bromoethyl)phthalimide, N-(3-bromopropyl)phthalimide, andallybromide. An exemplary electrophile is allylbromide.

The invention also includes methods of producing a compound of formula Iwhere at least two of R₁ through R₁₄ are (C1-C10) guanidoalkyloxy. Inone embodiment, a method includes contacting a compound of formula IV,where at least two of R₁ through R₁₄ are hydroxyl, with an electrophileto produce an alkyl ether compound of formula IV, where at least two ofR₁ through R₁₄ are (C1-C10)alkyloxy. The allyl ether compound isconverted into an amino precursor compound where at least two of R₁through R₁₄ are independently selected from the group consisting of(C1-C10) azidoalkyloxy and (C1-C10) cyanoalkyloxy. The amino precursorcompound is reduced to produce an aminoalkyl ether compound wherein atleast two of R₁ through R₁₄ are (C1-C10) aminoalkyloxy. The aminoalkylether compound is contacted with a guanidino producing electrophile toform a compound of formula I.

The term “guanidino producing electrophile” used herein refers to anelectrophile used to produce a guanidino compound of formula I. Anexample of an guanidino producing electrophile is HSO₃—C(NH)—NH₂.

The invention also includes methods of producing a compound of formula Iwhere at least two of R₁ through R₁₄ are H2N—HC(Q5)-C(O)—O— and Q5 isthe side chain of any amino acid. In one embodiment, a method includesthe step of contacting a compound of formula IV, where at least two ofR₁ through R₁₄ are hydroxyl, with a protected amino acid to produce aprotected amino acid compound of formula IV where at least two of atleast two of R₁ through R₁₄ are P.G.-HN—HC(Q5)-C(O)—O— and Q5 is theside chain of any amino acid and P.G. is an amino protecting group. Theprotecting group of the protected amino acid compound is removed to forma compound of formula I.

Exemplary non-limiting synthesis schemes for preparing compounds of theinvention include the following:

Compounds of the invention and precursors to the compounds according tothe invention are available commercially, e.g., from Sigm-Aldrich Co.,St. Louis; Mo.; and Research Plus, Inc., Manasquan, N.J. Other compoundsaccording to the invention can be synthesized according to methodsdisclosed herein, in U.S. Pat. Nos. 6,350,738; 6,486,148;and 6,767,904,and in the art.

Methods for identifying a candidate agent for treating a subject for apoxvirus infection or pathogenesis, for decreasing susceptibility of asubject to a poxvirus infection or pathogenesis, and decreasing orpreventing an adverse side effect caused by vaccination of a subjectwith or against a poxvirus, are provided. In one embodiment, a methodincludes providing a test agent, such as a cationic steroidantimicrobial (CSA); contacting the test agent with poxvirus andascertaining whether the test agent inhibits poxvirus infection orpathogenesis. A test agent identified as inhibiting poxvirus infectionor pathogenesis is a candidate agent for treating a subject for poxvirusinfection or pathogenesis. A test agent identified as inhibitingpoxvirus infection or pathogenesis is also a candidate agent fordecreasing susceptibility of a subject to a poxvirus infection orpathogenesis. A test agent identified as inhibiting poxvirus infectionor pathogenesis is further a candidate agent for decreasing orpreventing an adverse side effect caused by vaccination of a subjectwith a poxvirus. In various aspects, the subject is a mammal. Forexample, a mammal can comprises an animal model for poxvirus infectionor pathogenesis.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or study of the present invention, suitable methods andmaterials are described herein.

All of the features disclosed herein may be combined in any combination.Each feature disclosed in the specification may be replaced by analternative feature serving a same, equivalent, or similar purpose.Thus, unless expressly stated otherwise, disclosed features (e.g.,compound structures) are an example of a genus of equivalent or similarfeatures.

All applications, publications, patents and other references, GenBankcitations and ATCC citations cited herein are incorporated by referencein their entirety. In case of conflict, the specification, includingdefinitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a compound” includes a plurality of compounds andreference to “an anti-poxvirus effect, activity or function” can includereference to one or more effects, activities or functions, and so forth.

As used herein, all numerical values or numerical ranges includeintegers within such ranges and fractions of the values or the integerswithin ranges unless the context clearly indicates otherwise. Thus, toillustrate, reference to a range of 90-100%, includes 91%, 92%, 93%,94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%,etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. Referenceto a range of 0-72 hrs, includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as wellas 1, 2, 3, 4, 5, 6, 7 minutes, etc., and so forth. Reference to a rangeof 0-72 hrs, includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as well as 1, 2, 3,4, 5, 6, 7 minutes, etc., and so forth. Reference to a range of doses,such as 0.1-1 ug/kg, 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100 ug/kg,100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg,20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, includes0.11-0.9 ug/kg, 2-9 ug/kg, 11.5-24.5 ug/kg, 26-49 ug/kg, 55-90 ug/kg,125-400 ug/kg, 750-800 ug/kg, 1.1-4.9 mg/kg, 6-9 mg/kg, 11.5-19.5 mg/kg,21-49 mg/kg, 55-90 mg/kg, 125-200 mg/kg, 275.5-450.1 mg/kg, etc.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also includesembodiments in which subject matter is excluded, in full or in part,such as substances or materials, method steps and conditions, protocols,or procedures. Thus, even though the invention is generally notexpressed herein in terms of what the invention does not include aspectsthat are not expressly excluded in the invention are neverthelessdisclosed herein.

A number of embodiments of the invention have been described.Nevertheless, one skilled in the art, without departing from the spiritand scope of the invention, can make various changes and modificationsof the invention to adapt it to various usages and conditions. Forexample, salts, esters, ethers and amides of invention compoundsdisclosed herein are within the scope of this invention. Accordingly,the following examples are intended to illustrate but not limit thescope of invention described in the claims.

EXAMPLES

CSA compounds and intermediates were characterized using the followinginstruments: ¹H and ¹³C NMR spectra were recorded on a Varian Gemini2000 (200 MHz), Varian Unity 300 (300 MHz), or Varian VXR 500 (500 MHz)spectrometer and are referenced to TMS, residual CHCl₃ (¹H) or CDCl₃(¹³C), or residual CHD₂OD (¹H), or CD₃OD (¹³C). IR spectra were recordedon a Perkin Elmer 1600 FTIR instrument. Mass spectrometric data wereobtained on a JOEL SX 102A spectrometer. THF solvent was dried overNa/benzophenone and CH₂Cl₂ was dried over CaH₂ prior to use. Otherreagents and solvents were obtained commercially and were used asreceived.

Example 1

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 1-5, 13-20 and 22-27.

Compound 13: To a 1 L round-bottom flask were added methyl cholate(30.67 g, 72.7 mmol) in dry THF (600 mL) and LiAlH₄ (4.13 g, 109 mmol).After reflux for 48 hours, saturated aqueous Na₂SO₄ (100 mL) wasintroduced slowly, and the resulted precipitate was filtered out andwashed with hot THF and MeOH. Recrystallization from MeOH gave colorlesscrystals of 13 (28.0 g, 98% yield). m.p. 236.5-238° C.; IR (KBr) 3375,2934, 1373, 1081 cm⁻¹; ¹H NMR (CDCl₃/MeOH-d₄, 200 MHz) δ 3.98 (bs, 1 H),3.83 (bs, 1 H), 3.60-3.46 (m, 2 H), 3.38 (bs, 5 H), 2.30-2.10 (m, 2 H),2.05-1.05 (series of multiplets, 22 H), 1.03 (bs, 3 H), 0.92 (s, 3 H),0.71 (s, 3 H); ¹³C NMR (CDCl₃/MeOH-d₄, 50 MHz) δ 73.89, 72.44, 68.99,63.51, 48.05, 47.12, 42.49, 40.37, 39.99, 36.62, 36.12, 35.58, 35.40,32.77, 30.69, 30.04, 29.02, 28.43, 27.27, 23.96, 23.08, 18.00, 13.02;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 417.2992 (55.3%);calcd. 417.2981.

Compound 14: To a round-bottom flask were added 13 (28.2 g, 71.7 mmol)in DMF (300 ml), Et₃ N (20 mL, 143.4 mmol), trityl chloride (25.98 g,93.2 mmol) and DMAP (0.13 g, 1.07 mmol). The mixture was stirred at 50°C. under N₂ for 30 hours followed by the introduction of water (1000 mL)and extraction with EtOAc (5×200 mL). The combined extracts were washedwith water and brine and then dried over MgSO₄. After removal of solventin vacuo, the residue was purified using SiO₂ chromatography (CH₂Cl₂,Et₂O and MeOH as eluents) to give 14 as a pale yellow solid (31.9 g, 70%yield). m.p. 187° C. (decomposition); IR (KBr) 3405, 2935, 1448, 1075cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 7.46-7.42 (m, 6 H), 7.32-7.17 (m, 9 H),3.97 (bs, 1 H), 3.83 (bs, 1 H), 3.50-3.38 (m, 1 H), 3.01 (bs, 1 H), 2.94(dd, J=14.2, 12.2 Hz, 2 H), 2.64 (bs, 1 H), 2.51 (bs, 1 H), 2.36-2.10(m, 2 H), 2.00-1.05 (series of multiplets, 22 H), 0.96 (d, J=5.8 Hz, 3H), 0.87 (s, 3 H), 0.64 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 144.77,128.93, 127.91, 127.01, 86.43, 73.35, 72.06, 68.66, 64.28, 47.47, 46.53,41.74, 41.62, 39.64, 35.57, 35.46, 34.91, 34.82, 32.40, 30.55, 28.21,27.69, 26.80, 26.45, 23.36, 22.59, 17.83, 12.61; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 659.4069 (100%); calcd.659.4076.

Compound 15: To a round-bottom flask were added 14 (20.0 g, 31.4 mmol)in dry THF (600 mL) and NaH (60% in mineral oil, 6.3 g, 157.2 mmol). Themixture was refluxed for 30 min under N₂ followed by addition of allylbromide (27 mL, 314 mmol). After 60 hours of reflux, additional NaH (3eq.) and allyl bromide (4 eq.) were added. Following another 50 hours ofreflux, water (20 mL) was introduced slowly followed by addition of 1%HCl until the aqueous layer became neutral. The mixture was thenextracted with ether (3×100 mL) and the combined extracts were washedwith water (100 mL) and brine (2×100 mL). The ether solution was driedover anhydrous Na₂SO₄, and after removal of solvent, the residue waspurified using SiO₂ chromatography (hexanes and EtOAc/hexanes 1:8 aseluents) to give 15 (22.76 g, 96% yield) as a pale yellow glass. IR(neat) 2930, 1448, 1087 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 7.48-7.30 (m, 6H), 7.32-7.14 (m, 9 H), 6.04-5.80 (m, 3 H), 5.36-5.04 (series ofmultiplets, 6 H), 4.14-3.94 (m, 4 H), 3.74 (td, J=13.8, 5.8 Hz, 2 H),3.53 (bs, 1 H), 3.20-2.94 (m, 3 H), 3.31 (bs, 1 H), 2.38-1.90 (m, 4 H),1.90-0.96 (series of multiplets, 20 H), 0.90 (d, J=5.4 Hz, 3 H), 0.89(s, 3 H), 0.64 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 144.83, 136.27,136.08, 128.94, 127.90, 126.98, 116.46, 115.70, 86.42, 80.94, 79.29,74.98, 69.52, 69.39, 68.86, 64.39, 46.51, 46.42, 42.67, 42.14, 39.92,35.63, 35.51, 35.13, 32.45, 28.98, 28.09, 27.66, 27.57, 26.72, 23.32,23.11, 17.92, 12.69; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)779.5013 (86.1%); calcd. 779.5015.

Compound 16: To a three-necked round bottom flask was added 15 (3.34 g,4.4 mmol) in CH₂Cl₂ (200 mL) and methanol (100 mL). Through the coldsolution (−78° C.) ozone was bubbled through until a blue colorpersisted. Excess ozone was removed with oxygen flow. The mixture wasleft in a dry ice-acetone bath for an hour. Methyl sulfide (2.4 mL) wasadded and 15 minutes later, the mixture was treated with NaBH₄ (1.21 g,32 mmol) in 5% aqueous NaOH solution (10 mL)/methanol (10 mL) andallowed to warm to room temperature. The mixture was washed with brine(3×50 mL), and the combined brine wash was extracted with CH₂Cl₂ (2×50mL). The organic solution was dried over MgSO₄. After SiO₂chromatography (MeOH (5%) in CH₂ Cl₂), 3.30 g (95% yield) of 16 wasisolated as an oil. IR (neat) 3358, 2934, 1448, 1070 cm⁻¹; ¹H NMR(CDCl₃, 200 MHz) δ 7.50-7.42 (m, 6 H), 7.32-7.17 (m, 9 H), 3.80-2.96(series of multiplets, 20 H), 2.25-0.96 (series of multiplets, 24 H),0.89 (bs, 6 H), 0.65 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 144,73, 128.88,127.87, 126.96, 86.38, 81.05, 79.75, 76.59, 70.33, 69.66, 69.30, 64.20,62.25, 62.16, 62.03, 46.77, 46.36, 42.63, 41.77, 39.60, 35.43, 35.23,35.05, 34.89, 32.42, 28.91, 27.93, 27.56, 27.15, 26.68, 23.35, 22.98,22.85, 18.15, 12.60; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)791.4860 (100%), calcd. 791.4863.

Compound 17: To a round-bottom flask was added 16 (1.17 g, 1.55 mmol) indry THF (30 mL) under N₂ in ice-bath followed by 9-BBN/THF solution (0.5M, 10.2 mL, 5.51 mmol). The mixture was stirred at room temperature for12 hours. Aqueous NaOH (20%) (2 mL) and hydrogen peroxide (30%) (2 mL)were added in sequence. The mixture was refluxed for 1 hour followed bythe addition of brine (60 mL) and extraction with EtOAc (4×30 mL). Thecombined extracts were dried over anhydrous Na₂ SO₄. The product (1.01g, 80% yield) was obtained as a colorless oil after SiO₂ chromatography(5% MeOH in CH₂ Cl₂). IR (neat) 3396, 2936, 1448, 1365, 1089 cm⁻¹; ¹HNMR(CDCl₃, 200 MHz) δ 7.50-7.42 (m, 6 H), 7.34-7.16 (m, 9 H), 3.90-3.56(m, 13 H), 3.50 (bs, 1 H), 3.40-2.96 (series of multiplets, 6 H),2.30-0.94 (series of multiplets, 30 H), 0.90 (s, 3 H), 0.88 (d, J=5.4Hz, 3 H), 0.64 (s, 3 H); ¹³C NMR(CDCl₃, 50 MHz) δ 144.73, 128.88,127.85, 126.94, 86.36, 80.52, 78.90, 76.36, 66.82, 66.18, 65.77, 64.22,61.53, 61.41, 61.34, 46.89, 46.04, 42.60, 41.59, 39.60, 35.37, 35.27,34.88, 32.75, 32.44, 32.31, 28.82, 27.65, 27.48, 27.13, 26.77, 23.35,22.74, 22.38, 18.08, 12.48; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 833.5331 (100%), calcd. 833.5332.

Compound 18: To a round-bottom flask were added 16 (3.30 g, 4.29 mmol)in CH₂Cl₂ (150 mL) and NEt₃ (2.09 mL, 15.01 mmol). The mixture was putin ice-bath under N₂ followed by addition of mesyl chloride (1.10 mL,14.16 mmol). After 30 minutes, water (30 mL) and brine (200 mL) wereadded. The CH₂Cl₂ layer was washed with brine (2×50 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×100 mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The desired product (3.35 g, 78% yield) was isolatedas a pale yellow oil after SiO₂ chromatography (EtOAc/hexanes 1:1). IR(neat) 2937, 1448, 1352, 1174, 1120, 924 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ7.52-7.40 (m, 6 H), 7.34-7.20, (m, 9 H), 4.42-4.24 (m, 6 H), 3.90-3.64(m, 4 H), 3.60-3.30 (m, 4 H), 3.24-3.00 (m, 3 H), 3.10 (s, 6 H), 3.05(s, 3 H), 2.20-1.96 (m, 3 H)1.96-1.60 (m, 8 H), 1.60-0.94 (series ofmultiplets, 13 H), 0.91 (bs, 6 H), 0.65 (s, 3 H); ¹³C NMR(CDCl₃, 50 MHz)δ 114.68, 128.85, 127.85, 126.96, 86.37, 81.37, 79.58, 76.58, 69.95,69.43, 69.34, 66.52, 66.31, 65.59, 64.11, 46.80, 46.20, 42.65, 41.48,39.35, 37.82, 37.48, 35.36, 34.92, 34.73, 32.37, 28.66, 28.01, 27.44,27.03, 26.72, 23.17, 22.91, 22.72, 18.13, 12.50; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 1205.4176 (81.5%), calcd.1205.4189.

Compound 19: To a round-bottom flask were added 17 (1.01 g, 1.25 mmol)in CH₂Cl₂ (50 mL) and NEt₃ (0.608 mL, 4.36 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (0.318 mL, 4.11mmol). After 30 minutes, water (10 mL) and then brine (80 mL) wereadded. The CH₂ Cl₂ layer was washed with brine (2×20 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×40 mL). The combined extracts were washed with brine and dried overanhydrous Na₂ SO₄. The desired product (1.07 g, 82%) was isolated as apale yellowish oil after SiO₂ chromatography (EtOAc/hexanes 1:1). IR(neat) 2938, 1356, 1176, 1112 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.43,(m, 6 H), 7.32-7.22 (m, 9 H), 4.40-4.31 (m, 6 H), 3.72-3.64 (m, 2 H),3.55 (dd, J=6.3, 5.8 Hz, 2 H), 3.51 (bs, 1 H), 3.32-3.14 (m, 3 H),3.14-2.92 (m, 3 H), 3.01 (s, 3 H), 3.01 (s, 3 H), 3.00 (s, 3 H),2.10-1.92 (m, 10 H), 1.92-1.58 (m, 8 H), 1.56-0.92 (series ofmultiplets, 12 H), 0.90 (s, 3 H), 0.89 (d, J=5.4 Hz, 3 H), 0.64 (s, 3H); ¹³C NMR(CDCl₃, 75 MHz) δ 144.67, 128.85, 127.85, 126.96, 86.42,81.06, 79.83, 76.81, 68.12, 68.06, 68.02, 64.26, 64.06, 63.42, 46.76,46.38, 42.73, 41.87, 39.73, 37.44, 37.32, 37.29, 35.52, 35.48, 35.32,35.06, 32.53, 30.55, 30.28, 30.02, 29.15, 27.96, 27.69, 27.61, 26.75,23.52, 23.02, 18.17, 12.64; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 1067.4672 (100%), calcd. 1067.4659.

Compound 20: To a round-bottom flask were added 18 (1.50 g, 1.50 mmol)in dry DMSO (20 mL) and NaN₃ (0.976 g, 15 mmol). The mixture was heatedto 80° C. and stirred under N₂ overnight then diluted with water (100mL). The resulted aqueous mixture was extracted with EtOAc (3×50 mL),and the combined extracts washed with brine and dried over anhydrous Na₂SO₄. The desired product (0.83 g, 66% yield) was isolated as a clearglass after SiO₂ chromatography (EtOAc/hexanes 1:5). IR (neat) 2935,2106, 1448, 1302, 1114 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 7.50-7.42 (m, 6H), 7.36-7.20 (m, 9 H), 3.84-3.70 (m, 2 H), 3.65 (t, J=4.9 Hz, 2 H),3.55 (bs, 1 H), 3.44-3.08 (m, 10 H), 3.02 (t, J=6.4 Hz, 2 H), 2.38-0.96(series of multiplets, 24 H), 0.92 (d, J=5.6 Hz, 3 H), 0.91 (s, 3 H),0.65 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 114.84, 128.97, 127.92, 126.99,86.42, 81.24, 80.12, 76.59, 67.84, 67.29, 66.66, 64.36, 51.67, 51.44,51.18, 46.53, 46.23, 42.21, 41.93, 39.73, 35.66, 35.36, 35.06, 34.78,32.40, 28.95, 27.76, 27.39, 26.87, 23.45, 22.98, 22.92, 17.98, 12.53;HRFAB-MS (thioglycerol⁺Na⁺ matrix) m/e: ([M+Na]⁺) 866.5040 (100%),calcd. 866.5057.

Compound 22: To a round-bottom flask were added 20 (830 mg, 0.984 mmol)in MeOH (30 mL) and CH₂ Cl₂ (30 mL) and p-toluenesulfonic acid (9.35 mg,0.0492 mmol). The solution was stirred at room temperature for 2.5 hoursthen saturated aqueous NaHCO₃ (10 mL) was introduced. Brine (30 mL) wasadded, and the mixture was extracted with EtOAc (4×20 mL). The combinedextracts were dried over anhydrous Na₂ SO₄. The desired product (0.564g, 95% yield) was isolated as a pale yellowish oil after SiO₂chromatography (EtOAc/hexanes 1:2). IR (neat) 3410, 2934, 2106, 1301,1112 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 3.80-3.54 (m, 7 H), 3.44-3.20 (m,10 H), 2.35-0.96 (series of multiplets, 24 H), 0.95 (d, J=6.4 Hz, 3H),0.92 (s, 3 H), 0.68 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 81.10, 80.01,76.60, 67.75, 67.16, 66.56, 63.63, 51.57, 51.34, 51.06, 46.29, 46.12,42.12, 41.81, 39.60, 35.55, 35.23, 34.94, 34.66, 31.75, 29.48, 28.81,27.72, 27.66, 27.29, 23.32, 22.86, 22.80, 17.85, 12.39; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 624.3965 (100%), calcd.624.3962.

Compound 23: To a round-bottom flask were added 19 (1.07 g, 1.025 mmol)and NaN₃ (0.666 g, 10.25 mmol) followed the introduction of dry DMSO (15mL). The mixture was heated up to 80° C. under N₂ overnight. After theaddition of H₂ O (100 mL), the mixture was extracted with EtOAc (4×40mL) and the combined extracts were washed with brine (2×50 mL) and driedover anhydrous Na₂ SO₄. After removal of solvent, the residue wasdissolved in MeOH (15 mL) and CH₂ Cl₂ (15 mL) followed by the additionof catalytic amount of p-toluenesulfonic acid (9.75 mg, 0.051 mmol). Thesolution was stirred at room temperature for 2.5 hours before theaddition of saturated NaHCO₃ solution (15 mL). After the addition ofbrine (60 mL), the mixture was extracted with EtOAc (5×30 mL). Thecombined extracts were washed with brine (50 mL) and dried overanhydrous Na₂SO₄. The desired product (0.617 g, 94% yield for two steps)was obtained as a yellowish oil after SiO₂ chromatography (EtOAc/hexanes1:2). IR (neat) 3426, 2928, 2094, 1456, 1263, 1107 cm⁻¹; ¹H NMR (CDCl₃,300 MHz) δ 3.68-3.56 (m, 3 H), 3.56-3.34 (series of multiplets, 10 H),3.28-3.00 (series of multiplets, 4 H), 2.20-2.00 (m, 3 H), 1.98-1.55(series of multiplets, 15 H), 1.55-0.96 (series of multiplets, 13 H),0.92 (d, J=6.6 Hz, 3 H), 0.89 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (CDCl₃,75 MHz) δ 80.63, 79.79, 76.04, 64.99, 64.45, 64.30, 63.72, 49.01, 48.94,48.74, 46.49, 46.39, 42.70, 41.98, 39.80, 35.65, 35.42, 35.28, 35.08,31.99, 29.78, 29.75, 29.70, 29.49, 29.06, 27.87, 27.79, 27.65, 23.53,23.04, 22.85, 18.05, 12.59; HRFAB-MS (thioglycerol+Na matrix) m/e:([M+Na]⁺) 666.4415 (100%), calcd. 666.4431.

Compound 24: To a round-bottom flask were added 22 (0.564 g, 0.938 mmol)in CH₂Cl₂ (30 mL) and NEt₃ (0.20 mL, 1.40 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (0.087 mL, 1.13mmol). After 30 minutes, water (20 mL) and brine (100 mL) were added.The CH₂ Cl₂ layer was washed with brine (2×20 mL) and dried overanhydrous Na₂ SO₄. The combined aqueous mixture was extracted with EtOAc(3×30 mL). The combined extracts were washed with brine and dried overanhydrous Na₂ SO₄. The desired product (0.634 g, 99% yield) was isolatedas a pale yellowish oil after SiO₂ chromatography (EtOAc/hexanes 1:2).IR (neat) 2935, 2106, 1356, 1175, 1113 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ4.20 (t, J=6.8 Hz, 2 H), 3.80-3.75 (m, 1 H), 3.70-3.64 (m, 3 H), 3.55(bs, 1 H), 3.44-3.01 (m, 10 H), 3.00 (s, 3 H), 2.32-2.17 (m, 3 H),2.06-2.03 (m, 1 H), 1.90-0.88 (series of multiplets, 20 H), 0.95 (d,J=6.6 Hz, 3 H), 0.91 (s, 3 H), 0.68 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ80.90, 79.86, 76.43, 70.78, 67.64, 66.99, 66.48, 51.50, 51.26, 50.97,46.05, 45.96, 42.08, 41.71, 39.51, 37.33, 35.15, 34.86, 34.60, 31.34,28.73, 27.62, 27.59, 27.51, 25.68, 23.22, 22.80, 22.70, 17.62, 12.33;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 702.3741 (100%),calcd. 702.3737.

Compound 25: To a round-bottom flask were added 23 (0.617 g, 0.96 mmol)in CH₂ Cl₂ (30 mL) and NEt₃ (0.20 mL, 1.44 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (0.089 mL, 1.15mmol). After 30 minutes, water (20 mL) and brine (120 mL) were added.The CH₂ Cl₂ layer was washed with brine (2×20 mL) and dried overanhydrous Na₂ SO₄. The combined aqueous mixture was extracted with EtOAc(3×30 mL). The combined extracts were washed with brine and dried overanhydrous Na₂ SO₄. The desired product (0.676 g, 97% yield) was isolatedas a pale yellowish oil after removal of solvent. IR (neat) 2934, 2094,1454, 1360, 1174, 1112 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 4.17 (t, J=6.6Hz, 2 H), 3.65-3.28 (series of multiplets, 11 H), 3.64-3.00 (series ofmultiplets, 4 H), 2.97 (s, 3 H), 2.18-1.96 (series of multiplets, 16 H),1.54-0.94 (series of multiplets, 11 H), 0.89 (d, J=6.6 Hz, 3 H), 0.86(s, 3 H), 0.63 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.47, 79.67, 75.92,70.84, 64.90, 64.37, 64.17, 48.90, 48.86, 48.66, 46.32, 46.26, 42.63,41.87, 39.70, 37.39, 35.34, 35.28, 35.20, 34.99, 31.61, 29.68, 29.60,28.96, 27.78, 27.68, 27.57, 25.79, 23.41, 22.95, 22.74, 17.82, 12.50;HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺) 722.4385 (22.1%), calcd.722.4387.

Compound 26: To a 50 mL round-bottom flask was added 24 (0.634 g, 0.936mmol) and N-benzylmethylamine (2 mL). The mixture was heated under N₂ at80° C. overnight. Excess N-benzylmethylamine was removed under vacuum,and the residue was subjected to SiO₂ chromatography (EtOAc/hexanes1:2). The desired product (0.6236 g, 95% yield) was isolated as a paleyellow oil. IR (neat) 2935, 2106, 1452, 1302, 1116 cm⁻¹; ¹H NMR (CDCl₃,200 MHz) δ 7.32-7.24 (m, 5 H), 3.80-3.76 (m, 1 H), 3.70-3.60 (m, 3 H),3.54 (bs, 1 H), 3.47 (s, 2 H), 3.42-3.10 (m, 10 H), 2.38-2.05 (m, 5 H),2.17 (s, 3 H), 2.02-0.88 (series of multiplet, 21 H), 0.93 (d, J=7.0 Hz,3 H), 0.91 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 139.60,129.34, 128.38, 127.02, 81.22, 80.10, 76.71, 67.85, 67.29, 66.65, 62.45,58.38, 51.65, 51.44, 51.16, 46.50, 46.21, 42.40, 42.20, 41.93, 39.72,35.80, 35.34, 35.05, 34.76, 33.65, 28.93, 27082, 27.75, 27.38, 24.10,23.45, 22.98, 22.91, 18.05, 12.50; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M−H]⁺) 703.4748 (90.2%), calcd. 703.4772;([M+H]⁺) 705.4911(100%), calcd. 705.4928;([M+Na]⁺) 727.4767 (1.5%), calcd. 727.4748.

Compound 27: To a 50 mL round-bottom flask was added 25 (0.676 g, 0.937mmol) and N-benzylmethylamine (2 mL). The mixture was heated under N₂ at80° C. overnight. Excess N-benzylmethylamine was removed under vacuumand the residue was subjected to SiO₂ chromatography (EtOAc/hexanes1:2). The desired product (0.672 g, 96% yield) was isolated as a paleyellow oil. IR (neat) 2934, 2096, 1452, 1283, 1107 cm⁻¹; ¹H NMR (CDCl₃,300 MHz) δ 7.34-7.20 (m, 5 H), 3.68-3.37 (series of multiplets, 13 H),3.28-3.04 (m, 4 H), 2.33 (t, J=7.0 Hz, 2 H), 2.18 (s, 3 H), 2.20-2.00(m, 3 H), 1.96-1.56 (series of multiplets, 14 H), 1.54-1.12 (m, 10 H),1.10-0.96 (m, 3 H), 0.91 (d, J=8.7 Hz, 3 H), 0.89 (s, 3 H), 0.65 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 139.48, 129.23, 128.30, 126.96, 80.66,79.81, 76.08, 65.00, 64.46, 64.34, 62.50, 58.37, 49.02, 48.95, 48.75,46.65, 46.40, 42.69, 42.43, 42.00, 39.83, 35.86, 35.45, 35.30, 35.10,33.83, 29.81, 29.78, 29.72, 29.09, 27.88, 27.81, 27.66, 24.19, 23.57,23.06, 22.87, 18.15, 12.62; HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺)747.5406 (77.2%), calcd. 747.5398.

Compound 1: To a round-bottom flask were added 26 (0.684 g, 0.971 mmol)in dry THF (30 mL) and LiAlH₄ (113.7 mg, 3.0 mmol) under N₂. The mixturewas stirred at room temperature for 12 hours, and then Na₂SO₄.10 H₂Opowder (10 g) was added slowly. After the grey color disappeared, themixture was filtered through Celite and washed with dry THF. The product(0.581 g, 95% yield) was obtained as a colorless glass. IR (neat) 3372,2937, 1558, 1455, 1362, 1102 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.34-7.20(m, 5 H), 3.68-3.48 (m, 5 H), 3.47 (s, 2 H), 3.29 (bs, 1 H), 3.22-3.00(m, 3 H), 2.96-2.80 (m, 6 H), 2.32 (t, J=6.8, 5.4 Hz, 2 H), 2.17 (s, 3H), 2.20-2.00 (m, 3 H), 1.96-0.96 (series of multiplets, 27 H), 0.93 (d,J=6.8 Hz, 3 H), 0.90, (s, 3 H), 0.67 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ139.50, 129.22, 128.31, 126.96, 80.76, 79.85, 76.10, 70.90, 70.33,70.24, 62.48, 58.27, 46.55, 46.45, 42.72, 42.58, 42.33, 41.99, 39.77,35.78, 35.37, 35.01, 33.73, 29.07, 27.95, 27.71, 24.06, 23.46, 22.99,18.14, 12.55; HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺) 627.5211(100%), calcd. 627.5213.

HCl salt of compound 1: Compound 1 was dissolved in a minimum amount ofCH₂ Cl₂ and excess HCl in ether was added. Solvent and excess HCl wereremoved in vacuo and a noncrystalline white powder was obtained. ¹H NMR(methanol-d4/15% (CDCl₃, 300 MHz) δ 7.61-7.57 (m, 2 H), 7.50-7.48 (m, 3H), 4.84 (bs, 10 H), 4.45 (bs, 1 H), 4.30 (bs, 1 H), 3.96-3.82 (m, 2 H),3.78-3.69 (m, 2 H), 3.66 (bs, 1 H), 3.59-3.32 (series of multiplets, 4H), 3.28-3.02 (m, 8 H), 2.81 (s, 3 H), 2.36-2.15 (m, 4 H), 2.02-1.68 (m,8 H), 1.64-0.90 (series of multiplets, 12 H), 1.01 (d, J=6.35 Hz, 3 H),0.96 (s, 3 H), 0.73 (s, 3 H); ¹³ C NMR (methanol-d4/15% (CDCl₃, 75 MHz)δ 132.31, 131.20, 130.92, 130.40, 83.13, 81.09, 78.48, 65.54, 64.98,64.11, 60.87, 57.66, 47.51, 46.91, 43.52, 43.00, 41.38, 41.19, 41.16,40.75, 40.30, 36.37, 36.08, 36.00, 35.96, 33.77, 29.68, 29.34, 28.65,28.37, 24.42, 24.25, 23.33, 21.51, 18.80, 13.04.

Compound 2: To a round-bottom flask were added 27 (0.82 g, 1.10 mmol) indry THF (150 mL) and LiAlH₄ (125 mg, 3.30 mmol) under N₂. The mixturewas stirred at room temperature for 12 hours and Na₂ SO₄.10 H₂ O powder(10 g) was added slowly. After the grey color disappeared, the mixturewas filtered through a cotton plug and washed with dry THF. THF wasremoved in vacuo and the residue dissolved in CH₂ Cl₂ (50 mL). Afterfiltration, the desired product was obtained as a colorless glass (0.73g, 99% yield). IR (neat) 3362, 2936, 2862, 2786, 1576, 1466, 1363, 1103cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.23 (m, 5 H), 3.67-3.63 (m, 1 H),3.60-3.57 (m, 1H), 3.53 (t, J=6.4 Hz, 2 H), 3.47 (s, 2 H), 3.46 (bs, 1H), 3.24-3.17(m, 2 H), 3.12-2.99 (m, 2 H), 2.83-2.74 (series ofmultiplets, 6 H), 2.30 (t, J=7.3 Hz, 2 H), 2.15 (s, 3 H), 2.20-2.00 (m,3 H), 1.95-1.51 (series of multiplets, 20 H), 1.51-1.08, (series ofmultiplets, 10 H), 1.06-0.80 (m, 3 H), 0.87 (d, J=8.1 Hz, 3 H), 0.86 (s,3 H), 0.61 (s, 3 H); ¹³ C NMR (CDCl₃, 75 MHz).

139.35, 129.16, 128.22, 126.88, 80.44, 79.29, 75.96, 66.70, 66.52,66.12, 62.45, 58.26, 46.76, 46.27, 42.69, 42.41, 42.02, 40.68, 40.10,40.02, 39.82, 35.84, 35.47, 35.30, 35.06, 34.15, 34.09, 34.03, 33.80,28.96, 27.93, 27.75, 27.71, 24.32, 23.53, 23.03, 22.75, 18.17, 12.58;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 691.5504 (38.5%),calcd. 691.5502.

HCl salt of compound 2: Compound 2 was dissolved in a minimum amount ofCH₂ Cl₂ and excess HCl in ether was added. Removal of the solvent andexcess HCl gave a noncrystalline white powder. ¹H NMR (methanol-d₄/15%(CDCl₃, 300 MHz) δ 7.60-7.59 (m, 2 H), 7.50-7.47 (m, 3 H), 4.82 (bs, 10H), 4.43 (bs, 1 H), 4.32 (bs, 1 H), 3.85-3.79 (m, 1 H), 3.75-3.68 (m, 1H), 3.64 (t, J=5.74 Hz, 2 H), 3.57 (bs, 1 H), 3.36-3.28 (m, 2 H),3.25-3.00 (series of multiplets, 10 H), 2.82 (s, 3 H), 2.14-1.68 (seriesof multiplets, 19 H), 1.65-1.15 (series of multiplets, 11 H), 0.98 (d,J=6.6 Hz, 3 H), 0.95 (s, 3 H), 0.72 (s, 3 H); ¹³C NMR (methanol-d4/15%(CDCl₃, 75 MHz) δ 132.21, 131.10, 130.58, 130.28, 81.96, 80.72, 77.60,66.84, 66.58, 66.12, 61.03, 57.60, 44.16, 42.77, 40.62, 39.57, 39.43,36.28, 36.03, 35.96, 35.78, 33.65, 29.48, 29.27, 29.11, 29.01, 28.61,28.56, 28.35, 24.25, 23.56, 23.30, 21.17, 18.64, 12.90.

Compound 4: A suspension of 1 (79.1 mg, 0.126 mmol) andaminoiminomethanesulfonic acid (50.15 mg, 0.404 mmol) in methanol andchloroform was stirred at room temperature for 24 hours, and thesuspension became clear. An ether solution of HCl (1 M, 1 mL) was addedfollowed by the removal of solvent with N₂ flow. The residue wasdissolved in H₂ O (5 mL) followed by the addition of 20% aqueous NaOH(0.5 mL). The resulting cloudy mixture was extracted with CH₂Cl₂ (4×5mL). The combined extracts were dried over anhydrous Na₂SO₄. Removal ofsolvent gave the desired product (90 mg, 95%) as white powder. m.p.111-112° C. IR (neat) 3316, 2937, 1667, 1650, 1556, 1454, 1348, 1102cm⁻¹; ¹H NMR (5% methanol-d4/CDCl₃, 300 MHz) δ 7.26-7.22 (m, 5 H), 4.37(bs, 3 H), 3.71-3.51(series of multiplets, 5 H), 3.44 (s, 2 H),3.39-3.10 (series of multiplets, 10 H), 2.27 (t, J=6.83 Hz, 2 H), 2.13(s, 3 H), 2.02-0.94 (series of multiplets, 33 H), 0.85 (d, J=5.62 Hz, 3H), 0.84 (s, 3 H), 0.61 (s, 3 H); ¹³C NMR (5% methanol-d4/CDCl₃, 75 MHz)δ 158.54, 158.48, 158.43, 138.27, 129.47, 128.32, 127.19, 81.89, 80.30,77.34, 69.02, 68.46, 67.21, 62.36, 58.00, 47.36, 46.18, 43.26, 43.00,42.73, 42.18, 41.48, 39.32, 35.55, 34.97, 34.89, 34.67, 33.63, 28.93,28.28, 27.53, 27.16, 23.96, 23.28, 23.16, 22.77, 18.36, 12.58; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 753.5858 (100%), calcd.753.5867.

HCl salt of compound 4: Compound 4 was dissolved in minimum amount ofCH₂Cl₂ and MeOH followed by addition of excess HCl in ether. The solventwas removed by N₂ flow, and the residue was subjected to high vacuumovernight. The desired product was obtained as noncrystalline whitepowder. ¹H NMR (methanol-d4/20% (CDCl₃, 300 MHz) δ 7.58 (bs, 2 H),7.50-7.48 (m, 3 H), 4.76 (bs, 13 H), 4.45 (d, J=12.9 Hz, 1 H), 4.27 (dd,1 H, J=12.9, 5.4 Hz), 3.82-3.00 (series of multiplets, 17 H), 2.81-2.80(m, 3 H), 2.20-1.02 (series of multiplets, 27 H), 0.98 (d, J=6.59 Hz, 3H), 0.95 (s, 3 H), 0.72 (s, 3 H); ¹³C NMR (methanol-d4/20% CDCl₃, 75MHz) δ 158.88, 158.72, 132.00, 131.96, 130.98, 130.15, 82.51, 81.07,78.05, 68.50, 68.02, 67.94, 67.10, 60.87, 60.53, 57.38, 47.16, 46.91,43.91, 43.11, 43.01, 42.91, 42.55, 40.28, 39.88, 39.95, 35.90, 35.73,35.64, 33.53, 29.18, 28.35, 27.99, 24.02, 23.30, 21.35, 18.52, 18.44,13.06.

Compound 5: A suspension of 2 (113 mg, 0.169 mmol) andaminoiminomethanesulfonic acid (67.1 mg, 0.541 mmol) in methanol andchloroform was stirred at room temperature for 24 hours. HCl in ether (1M, 1 mL) was added followed by the removal of solvent with N₂ flow. Theresidue was subject to high vacuum overnight and dissolved in H₂ O (5mL) followed by the addition of 20% NaOH solution (1.0 mL). Theresulting mixture was extracted with CH₂ Cl₂ (5×5 mL). The combinedextracts were dried over anhydrous Na₂ SO₄. Removal of solvent gavedesired the product (90 mg, 95% yield) as a white solid. m.p. 102-104°C. IR (neat) 3332, 3155, 2939, 2863, 1667, 1651, 1558, 1456, 1350, 1100cm⁻¹; ¹ H NMR (5% methanol-d4/CDCl₃, 300 MHz) δ 7.35-7.24 (m, 5 H),3.75-3.64 (m, 1 H), 3.57 (bs, 5 H), 3.50 (s, 2 H), 3.53-3.46 (m, 1 H),3.40-3.10 (series of multiplets, 14 H), 2.34 (t, J=7.31 Hz, 2 H), 2.19(s, 3 H), 2.13-0.96 (series of multiplets, 36 H), 0.91 (bs, 6 H), 0.66(s, 3 H); ¹³C NMR (5% methanol-d4/CDCl₃, 75 MHz) δ 157.49, 157.31,157.23, 138.20, 129.52, 128.34, 127.23, 81.17, 79.19, 76.42, 65.63,65.03, 64.70, 62.36, 58.02, 47.23, 46.24, 42.89, 42.18, 41.45, 39.45,39.40, 39.30, 38.71, 35.61, 35.55, 35.02, 34.82, 33.69, 29.87, 29.59,29.42, 28.84, 27.96, 27.56, 23.95, 23.40, 22.82, 22.64, 18.28, 12.54;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 795.6356 (84.3%),calcd. 795.6337.

HCl salt of compound 5: Compound 5 was dissolved in minimum amount ofCH₂ Cl₂ and MeOH followed by the addition of excess HCl in ether. Thesolvent and excess HCl were removed by N₂ flow and the residue wassubject to high vacuum overnight. The desired product was obtained asnoncrystalline white powder. ¹ H NMR (methanol-d4/10% CDCl₃, 300 MHz) δ7.62-7.54 (m, 2 H), 7.48-7.44 (m, 3 H), 4.84 (bs, 16 H), 4.46 (d, J=12.7Hz, 1 H), 4.26 (dd, J=12.7, 3.42 Hz, 1 H), 3.78-3.56 (series ofmultiplets, 5 H), 3.38-3.05 (series of multiplets, 13 H), 2.80 (d, 3 H),2.19-2.04 (m, 3 H), 2.02-1.04 (series of multiplets, 30 H), 0.98 (d,J=6.35 Hz, 3 H), 0.95 (s, 3 H), 0.72 (s, 3 H); ¹³C NMR (methanol-d4/10%CDCl₃, 75 MHz) δ 158.75, 158.67, 132.32, 131.24, 130.83, 130.43, 82.49,81.02, 77.60, 66.47, 65.93, 61.19, 60.85, 57.69, 47.79, 47.60, 44.29,43.07, 40.86, 40.42, 40.19, 40.09, 39.76, 36.68, 36.50, 36.15, 35.94,33.91, 30.75, 30.46, 29.74, 29.33, 28.71, 24.41, 24.03, 23.38, 22.21,22.16, 18.59, 18.52, 13.09.

Compound CSA-26 was synthesized according to Scheme 1 and Example 1using 7-deoxycholic acid in place of cholic acid and methyl cholate.

Example 2

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 3, 28 and 29.

Compound 28: A suspension of 19 (0.641 g, 0.614 mmol) and KCN (0.40 g,6.14 mmol) in anhydrous DMSO (5 mL) was stirred under N₂ at 80° C.overnight followed by the addition of H₂ O (50 mL). The aqueous mixturewas extracted with EtOAc (4×20 mL). The combined extracts were washedwith brine once, dried over anhydrous Na₂ SO₄ and concentrated in vacuo.The residue was dissolved in CH₂ Cl₂ (3 mL) and MeOH (3 mL) andcatalytic amount of p-toluenesulfonic acid (5.84 mg, 0.03 mmol) wasadded. The solution was stirred at room temperature for 3 hours beforethe introduction of saturated NaHCO₃ solution (10 mL). After theaddition of brine (60 mL), the mixture was extracted with EtOAc (4×30mL). The combined extracts were washed with brine once and dried overanhydrous Na₂ SO₄ and concentrated. The residue afforded the desiredproduct (0.342 g, 92% yield) as pale yellowish oil after columnchromatography (silica gel, EtOAc/hexanes 2:1). IR (neat) 3479, 2936,2864, 2249, 1456, 1445, 1366, 1348, 1108 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ3.76-3.53 (m, 7 H), 3.32-3.06 (series of multiplets, 4 H), 2.57-2.46 (m,6 H), 2.13-1.00 (series of multiplets, 31 H), 0.93 (d, J=6.35 Hz, 3 H),0.90 (s, 3 H), 0.67 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 119.91, 119.89,80.75, 79.65, 76.29, 65.83, 65.37, 65.19, 63.63, 46.57, 46.44, 42.77,41.79, 39.71, 35.63, 35.26, 35.02, 32.00, 29.46, 29.03, 27.96, 27.74,26.64, 26.42, 26.12, 23.56, 22.98, 22.95, 18.24, 14.65, 14.54, 14.30,12.60; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 618.4247(67.8%), calcd. 618.4247.

Compound 29: To a solution of 28 (0.34 g, 0.57 mmol) in dry CH₂ Cl₂ (15mL) under N₂ at 0° C. was added NEt₃ (119.5 μL, 0.857 mmol) followed bythe addition of mesyl chloride (53.1 .mu.L, 0.686 mmol). The mixture wasallowed to stir at 0° C. for 30 minutes before the addition of H₂ O (6mL). After the introduction of brine (60 mL), the aqueous mixture wasextracted with EtOAc (4×20 mL). The combined extracts were washed withbrine once, dried over anhydrous Na₂ SO₄ and concentrated. To theresidue was added N-benzylmethyl amine (0.5 mL) and the mixture wasstirred under N₂ at 80° C. overnight. Excess N-benzylmethylamine wasremoved in vacuo and the residue was subject to column chromatography(silica gel, EtOAc/hexanes 2:1 followed by EtOAc) to afford product(0.35 g, 88% yield) as a pale yellow oil. IR (neat) 2940, 2863, 2785,2249, 1469, 1453, 1366, 1348, 1108 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ7.34-7.21 (m, 5 H), 3.76-3.69 (m, 1 H), 3.64-3.50 (m, 4 H), 3.48 (s, 2H), 3.31-3.05 (series of multiplets, 4 H), 2.52-2.46 (m, 6 H), 2.33 (t,J=7.32 H, 2 Hz), 2.18 (s, 3 H), 2.13-0.95 (series of multiplets, 30 H),0.91 (d, J=6.80 H, 3 Hz), 0.90 (s, 3 H), 0.66 (s, 3 H); ¹³ C NMR (CDCl₃,75 MHz) δ 139.37, 129.17, 128.26, 126.93, 119.96, 119.91, 80.73, 79.59,76.26, 65.79, 65.35, 65.13, 62.47, 58.25, 46.74, 46.40, 42.72, 42.38,41.76, 39.68, 35.78, 35.22, 34.98, 33.79, 28.99, 27.92, 27.71, 26.63,26.38, 26.09, 24.21, 23.54, 22.96, 22.90, 18.28, 14.62, 14.51, 14.26,12.58; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 699.5226 (100%),calcd. 699.5213.

Compound 3: A solution of 29 (0.074 g, 0.106 mmol) in anhydrous THF (10mL) was added dropwise to a mixture of AlCl₃ (0.1414 g, 1.06 mmol) andLiAlH₄ (0.041 g, 1.06 mmol) in dry THF (10 mL). The suspension wasstirred for 24 hours followed by the addition of 20% NaOH aqueoussolution (2 mL) at ice-bath temperature. Anhydrous Na₂ SO₄ was added tothe aqueous slurry. The solution was filtered and the precipitate washedtwice with THF. After removal of solvent, the residue was subject tocolumn chromatography (silica gel, MeOH/CH₂ Cl₂ 1:1 followed by MeOH/CH₂Cl₂/NH₃.H₂ O 4:4:1) to afford the desired product (0.038 g, 50% yield)as a clear oil. IR (neat) 3362, 2935, 2863, 2782, 1651, 1574, 1568,1557, 1471, 1455, 1103 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.22 (m, 5H), 3.60-3.02 (series of broad multiplets, 18 H), 2.90-2.70 (m, 5 H),2.33 (t, J=7.20 Hz, 2 H), 2.24-2.04 (m, 3 H), 2.18 (s, 3 H), 1.96-0.96(series of multiplets, 30 H), 0.90 (d, J=7.57 Hz, 3 H), 0.89 (s, 3 H),0.64 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 139.44, 129.24, 128.31, 126.97,80.63, 79.65, 75.97, 68.44, 68.00, 67.96, 62.54, 58.40, 46.77, 46.30,42.73, 42.43, 42.07, 41.92, 41.74, 41.72, 39.81, 35.82, 35.48, 35.07,33.84, 31.04, 30.30, 30.10, 29.03, 28.11, 27.82, 27.81, 27.74, 27.67,27.64, 24.31, 23.50, 23.04, 22.93, 18.22, 12.63; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 711.6139 (100%), calcd.711.6152;([M+Na]⁺) 733.5974 (46.1%), calcd. 733.5972.

Example 3

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 6, 7 and 30-33.

Compound 30: Cholic acid (3.0 g, 7.3 mmol) was dissolved in CH₂Cl₂ (50mL) and methanol (5 mL). Dicyclohexylcarbodiimide (DCC) (1.8 g, 8.8mmol) was added followed by N-hydroxysuccinimide (about 100 mg) andbenzylmethylamine (1.1 g, 8.8 mmol). The mixture was stirred for 2hours, then filtered. The filtrate was concentrated and chromatographed(SiO₂, 3% MeOH in CH₂Cl₂) to give 3.0 g of a white solid (81% yield).m.p. 184-186° C.; IR (neat) 3325, 2984, 1678 cm⁻¹; ¹H NMR (CDCl₃, 200MHz) δ 7.21 (m, 5 H), 4.51 (m, 2 H), 3.87 (m, 1 H), 3.74 (m, 2 H), 3.36(m, 2 H), 2.84 (s, 3 H), 2.48-0.92 (series of multiplets, 28 H), 0.80(s, 3 H), 0.58 (d, J=6.5 Hz, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 174.30,173.94, 137.36, 136.63, 128.81, 128.46, 127.85, 127.50, 127.18, 126.28,72.96, 71.76, 68.35, 53.39, 50.65, 48.77, 46.91, 46.33, 41.44, 39.36,39.18, 35.76, 35.27, 34.76, 33.87, 31.54, 34.19, 31.07, 30.45, 28.11,27.63, 26.14, 25.59, 24.92, 23.26, 17.51, 12.41; FAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 512 (100%), calcd. 512.

Compound 31: Compound 30 (2.4 g, 4.7 mmol) was added to a suspension ofLiAlH₄ (0.18 g, 4.7 mmol) in THF (50 mL). The mixture was refluxed for24 hours, then cooled to 0° C. An aqueous solution of Na₂SO₄ wascarefully added until the grey color of the mixture dissipated. Thesalts were filtered out, and the filtrate was concentrated in vacuo toyield 2.1 g of a white solid (88%). The product proved to be ofsufficient purity for further reactions. m.p. 70-73° C.; IR (neat) 3380,2983, 1502 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.23 (m, 5 H), 3.98 (bs, 2H), 3.81 (m, 3 H), 3.43 (m, 3 H), 2.74 (m, 2 H), 2.33 (m, 3 H), 2.25 (s,3 H), 2.10-0.90 (series of multiplets, 24 H), 0.98 (s, 3 H), 0.78 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 135.72, 129.63, 128.21, 128.13, 125.28,72.91, 71.63, 62.05, 60.80, 56.79, 47.00, 46.23, 41.44, 40.81, 39.41,35.42, 35.24, 34.63, 34.02, 33.22, 31.73, 30.17, 29.33, 29.16, 28.02,27.49, 26.17, 25.55, 23.10, 22.48, 22.33, 17.54, 12.65; FAB-MS(thioglycerol matrix) m/e: ([M+H]⁺) 498 (100%), calcd. 498.

Compound 32: Compound 31 (0.36 g, 0.72 mmol) was dissolved in CH₂Cl₂ (15mL) and Bocglycine (0.51 g, 2.89 mmol), DCC (0.67 g, 3.24 mmol) anddimethylaminopyridine (DMAP) (about 100 mg) were added. The mixture wasstirred under N₂ for 4 hours then filtered. After concentration andchromatography (SiO₂, 5% MeOH in CH₂Cl₂), the product was obtained as a0.47 g of a clear glass (68%). ¹H NMR (CDCl₃, 300 MHz) δ 7.30 (m, 5 H),5.19 (bs, 1 H), 5.09 (bs, 3 H), 5.01 (bs, 1 H), 4.75 (m, 1 H), 4.06-3.89(m, 6 H), 2.33 (m, 2 H), 2.19 (s, 3 H) 2.05-1.01 (series of multiplets,26 H), 1.47 (s, 9 H), 1.45 (s, 18 H), 0.92 (s, 3 H), 0.80 (d, J=6.4 Hz,3 H), 0.72 (s, 3 H). ³C NMR (CDCl₃, 75 MHz) δ 170.01, 169.86, 169.69,155.72, 155.55, 139.90, 129.05, 128.17, 126.88, 79.86, 76.53, 75.09,72.09, 62, 35, 57.88, 47.78, 45.23, 43.12, 42.79, 42.16, 40.81, 37.94,35.51, 34.69, 34.57, 34.36, 33.30, 31.31, 29.66, 28.80, 28.34, 27.22,26.76, 25.61, 24.02, 22.83, 22.47, 17.93, 12.19; FAB-MS (thioglycerolmatrix) m/e: ([M+H]⁺) 970 (100%), calcd. 970.

Compound 33: Compound 31 (0.39 g, 0.79 mmol) was dissolved in CH₂Cl₂ (15mL) and Boc-β-alanine (0.60 g, 3.17 mmol), DCC (0.73 g, 3.56 mmol) anddimethylaminopyridine (DMAP) (about 100 mg) were added. The mixture wasstirred under N₂ for 6 hours then filtered. After concentration andchromatography (SiO₂, 5% MeOH in CH₂Cl₂), the product was obtained as a0.58 g of a clear glass (72%). IR (neat) 3400, 2980, 1705, 1510 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ 7.27 (m, 5 H), 5.12 (bs, 4 H), 4.93 (bs, 1 H),4.71 (m, 1 H), 3.40 (m, 12 H), 2.59-2.48 (m, 6 H), 2.28 (m, 2 H), 2.17(s, 3 H), 2.05-1.01 (series of multiplets, 26 H), 1.40 (s, 27 H), 0.90(s, 3 H), 0.77 (d, J=6.1 Hz, 3 H), 0.70 (s, 3 H). ¹³C NMR (CDCl₃, 75MHz) δ 171.85, 171.50, 171.44, 155.73, 138.62, 129.02, 128.09, 126.87,79.18, 75.53, 74.00, 70.91, 62.20, 57.67, 47.84, 44.99, 43.28, 41.98,40.73, 37.67, 36.12, 34.94, 34.65, 34.47, 34.20, 33.29, 31.23, 29.57,28.74, 28.31, 28.02, 27.86, 27.12, 26.73, 25.46, 24.86, 23.95, 22.77,22.39, 17.91, 12.14; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺)1011.6619 (100%), calcd. 1011.6634.

Compound 6: Compound 32 (0.15 g, 0.15 mmol) was stirred with excess 4 NHCl in dioxane for 40 minutes. The dioxane and HCl were removed in vacuoleaving 0.12 g of a clear glass (about 100%). ¹H NMR (CD₃OD, 300 MHz) δ7.62 (bs, 2 H), 7.48 (bs, 3 H), 5.30 (bs, 1 H), 5.11 (bs, 1 H), 4.72 (bs(1 H), 4.46 (m, 1 H), 4.32 (m, 1H) 4.05-3.91 (m, 4 H), 3.10 (m, 2 H),2.81 (s, 3 H), 2.15-1.13 (series of multiplets, 25 H), 1.00 (s, 3 H),0.91 (bs, 3 H), 0.82 (s, 3 H). ¹³C NMR (CD₃OD, 125 MHz) δ 166.86,166.50, 131.09, 130.18, 129.17, 128.55, 76.60, 75,43, 72.61, 72.04,70.40, 66.22, 60.07, 58.00, 57.90, 54.89, 54.76, 46.44, 44.64, 43.39,42.22, 38.56, 36.78, 34.14, 33.92, 33.84, 31.82, 30.54, 29.67, 28.79,27.96, 26.79, 26.00, 24.99, 23.14, 22.05, 21.82, 19.91, 17.27, 11.60;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M-4 Cl-3 H]⁺) 669.4576 (100%),calcd. 669.4591.

Compound 7: Compound 33 (0.20 g, 0.20 mmol) was stirred with excess 4 NHCl in dioxane for 40 minutes. The dioxane and HCl were removed in vacuoleaving 0.12 g of a clear glass (about 100%). ¹H NMR (CD₃OD, 500 MHz) δ7.58 (bs, 2 H), 7.49 (bs, 3 H), 5.21 (bs, 1 H), 5.02 (bs, 1 H), 4.64 (m,1 H), 4.44 (m, 1 H), 4.28 (m, 1 H), 3.30-2.84 (m, 14 H), 2.80 (s, 3 H),2.11-1.09 (series of multiplets, 25 H), 0.99 (s, 3 H), 0.89 (d, J=4.1Hz, 3 H), 0.80 (s, 3 H); ¹³ C NMR (CD₃ OD, 125 MHz) δ 171.92, 171.56,171.49, 132.44, 131.32, 131.02, 130.51, 78.13, 76.61, 61.45, 57.94,46.67, 44.80, 42.36, 40.85, 39.33, 37.03, 36.89, 36.12, 36.09, 35.79,35.63, 33.81, 33.10, 32.92, 32.43, 30.28, 28.43, 28.04, 26.65, 24.02,22.86, 21.98, 18.70, 12.68; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M-4 Cl-3 H]⁺) 711.5069 (43%), calcd. 711.5061.

Example 4

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 8, CSA-7, CSA-8 and 34-40.

Compound 34: Diisopropyl azodicarboxylate (DIAD) (1.20 mL, 6.08 mmol)was added to triphenylphosphine (1.60 g, 6.08 mmol) in THF (100 mL) at0° C. and was stirred for half an hour during which time the yellowsolution became a paste. Compound 14 (2.58 g, 4.06 mmol) andp-nitrobenzoic acid (0.81 g, 4.87 mmol) were dissolved in THF (50 mL)and added to the paste. The resulted mixture was stirred at ambienttemperature overnight. Water (100 mL) was added and the mixture was madeslightly basic by adding NaHCO₃ solution followed by extraction withEtOAc (3×50 mL). The combined extracts were washed with brine once anddried over anhydrous Na₂ SO₄. The desired product (2.72 g, 85% yield)was obtained as white powder after SiO₂ chromatography (Et₂ O/hexanes1:2). m.p. 207-209° C.; IR (KBr) 3434, 3056, 2940, 2868, 1722, 1608,1529, 1489, 1448, 1345 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.30-8.26 (m, 2H), 8.21-8.16 (m, 2 H), 7.46-7.42 (m, 6 H), 7.31-7.18 (m, 9 H)5.33 (bs,1 H), 4.02 (bs, 1 H), 3.90 (bs, 1 H), 3.09-2.97 (m, 2 H), 2.68 (td,J=14.95, 2.56 Hz, 1 H), 2.29-2.19 (m, 1 H), 2.07-1.06 (series ofmultiplets, 24 H), 1.01 (s, 3 H), 0.98 (d, J=6.6 Hz, 3 H), 0.70 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 164.21, 150.56, 144.70, 136.79, 130.77,128.88, 127.86, 126.98, 123.70, 86.47, 73.24, 73.00, 68.70, 64.22,47.79, 46.79, 42.15, 39.76, 37.47, 35.52, 35.34, 34.23, 33.79, 32.46,31.12, 28.74, 27.71, 26.85, 26.30, 25.16, 23.41, 17.98, 12.77; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 808.4203 (53.8%), calcd.808.4189. Nitrobenzoate (2.75 g, 3.5 mmol) was dissolved in CH₂Cl₂ (40mL) and MeOH (20 mL) and 20% aqueous NaOH (5 mL) were added. The mixturewas heated up to 60° C. for 24 hours. Water (100 mL) was introduced andextracted with EtOAc. The combined extracts were washed with brine anddried over anhydrous Na₂ SO₄. The desired product (1.89 g, 85% yield)was obtained as white solid after SiO₂ chromatography (3% MeOH in CH₂Cl₂ as eluent). m.p. 105-106° C.; IR (KBr) 3429, 3057, 2936, 1596, 1489,1447, 1376, 1265, 1034, 704 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42(m, 6 H), 7.32-7.19 (m, 9 H), 4.06 (bs, 1 H), 3.99 (bs, 1 H), 3.86 (bd,J=2.44 Hz, 1 H), 3.09-2.97 (m, 2 H), 2.47 (td, J=14.03, 2.44 Hz, 1 H),2.20-2.11 (m, 1 H), 2.04-1.04 (series of multiplets, 25 H), 0.97 (d,J=6.59 Hz, 3 H), 0.94 (s, 3 H), 0.68 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ144.70, 128.88, 127.86, 126.97, 86.45, 73.31, 68.84, 67.10, 64.23,47.71, 46.74, 42.10, 39.70, 36.73, 36.73, 36.15, 35.53, 35.45, 34.45,32.46, 29.93, 28.67, 27.86, 27.71, 26.87, 26.04, 23.43, 23.16, 17.94,12.75; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 659.4064(100%), calcd. 659.4076.

Compound 35: To a round-bottom flask were added 34 (2.0 g, 3.14 mmol),NaH (60% in mineral oil, 3.8 g, 31.4 mmol) and THF (150 mL). Thesuspension was refluxed for 2 hours followed by the addition of allylbromide (2.72 mL, 31.4 mL). After refluxing for 28 hours, another 10 eq.of NaH and allyl bromide were added. After 72 hours, another 10 eq. ofNaH and allyl bromide were added. After 115 hours, TLC showed almost nostarting material or intermediates. Water (100 mL) was added to thesuspension carefully, followed by extraction with EtOAc (5×50 mL). Thecombined extracts were washed with brine and dried over anhydrousNa₂SO₄. The desired product (1.81 g, 79% yield) was obtained as ayellowish glass after SiO₂ chromatography (5% EtOAc/hexanes). IR (neat)3060, 3020, 2938, 2865, 1645, 1596, 1490, 1448, 1376, 1076, 705 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6 H), 7.31-7.18 (m, 9 H), 6.06-5.85(m, 3 H), 5.35-5.20 (m, 3 H), 5.15-5.06 (m, 3 H), 4.10-4.00 (m, 2 H),3.93-3.90 (m, 2 H), 3.85-3.79 (ddt, J=13.01, 4.88, 1.59 Hz, 1 H),3.73-3.66 (ddt, J=13.01, 5.38, 1.46 Hz, 1 H), 3.58 (bs, 1 H), 3.54 (bs,1 H), 3.32 (d, J=2.93 Hz, 1 H), 3.07-2.96 (m, 2 H), 2.36 (td, J=13.67,2.68 Hz, 1 H), 2.24-2.10 (m, 2 H), 2.03-1.94 (m, 1 H), 1.87-0.86 (seriesof multiplets, 20 H), 0.91 (s, 3 H), 0.90 (d, J=6.83 Hz, 3 H), 0.64 (s,3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.77, 136.29, 136.21, 136.13, 128.90,127.86, 126.94, 116.13, 115.51, 115.42, 86.44, 81.11, 75.65, 73.92,69.40, 68.81, 64.43, 46.68, 46.54, 42.93, 39.93, 36.98, 35.66, 35.14,35.14, 32.83, 32.54, 30.48, 28.51, 27.72, 27.64, 26.82, 24.79, 23.65,23.43, 23.40, 18.07, 12.80; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+H]⁺) 757.5185 (12.9%), calcd. 757.5196.

Compound 36: Ozone was bubbled through a solution of 35 (0.551 g, 0.729mmol) in CH₂ Cl₂ (40 mL) and MeOH (20 mL) at −78° C. until the solutionturned a deep blue. Excess ozone was blown off with oxygen.Methylsulfide (1 mL) was added followed by the addition of NaBH₄ (0.22g, 5.80 mmol) in 5% NaOH solution and methanol. The resulted mixture wasstirred overnight at room temperature and washed with brine. The brinewas then extracted with EtOAc (3×20 mL). The combined extracts weredried over Na₂ SO₄. The desired product (0.36 g, 65% yield) was obtainedas a colorless glass after SiO₂ chromatography (5% MeOH/CH₂ Cl₂). IR(neat) 3396, 3056, 2927, 1596, 1492, 1462, 1448, 1379, 1347, 1264, 1071cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6 H), 7.32-7.18 (m, 9 H),3.77-3.57 (series of multiplets, 10 H), 3.48-3.44 (m, 2 H), 3.36-3.30(m, 2 H), 3.26-3.20 (m, 1 H), 3.04-2.99 (m, 2 H), 2.37-0.95 (series ofmultiplets, 27 H), 0.92 (s, 3 H), 0.91 (d, J=6.59 Hz, 3 H), 0.67 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.69, 128.87, 127.84, 126.94, 86.44,81.05, 76.86, 74.65, 69.91, 69.22, 68.77, 64.24, 62.44, 62.42, 62.26,46.92, 46.54, 42.87, 39.73, 36.86, 35.52, 35.13, 32.82, 32.54, 30.36,28.71, 27.61, 27.44, 26.79, 24.82, 23.51, 23.38, 23.31, 18.28, 12.74;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 791.4844 (96.4%),calcd. 791.4863.

Compound 37: NEt₃ (0.23 mL, 1.66 mmol) was added to a solution of 36(0.364 g, 0.47 mmol) in dry CH₂ Cl₂ (30 mL) at 0° C. under N₂ followedby the introduction of mesyl chloride (0.12 mL, 1.56 mmol). The mixturewas stirred for 10 minutes and H₂ O (10 mL) added to quench thereaction, followed by extraction with EtOAc (3×30 mL). The combinedextracts were washed with brine and dried over anhydrous Na₂ SO₄. SiO₂chromatography (EtOAc/hexanes 1:1) gave the desired product (0.411 g,86% yield) as white glass. IR (neat) 3058, 3029, 2939, 2868, 1491, 1461,1448, 1349, 1175, 1109, 1019 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42(m, 6 H), 7.31-7.19 (m, 9 H), 4.35-4.26 (m, 6 H), 3.84-3.74 (m, 2 H),3.64-3.56 (m, 4 H), 3.49-3.34 (m, 3 H), 3.06 (s, 3 H), 3.04 (s, 3 H),3.02 (s, 3 H), 3.09-2.95 (m, 2 H), 2.28 (bt, J=14.89 Hz, 1 H), 2.09-0.86(series of multiplets, 21 H), 0.92 (s, 3 H), 0.90 (d, J=6.78 Hz, 3 H),0.66 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.66, 128.86, 127.86, 126.97,86.46, 81.28, 77.18, 75.00, 70.14, 69.89, 69.13, 66.49, 65.85, 65.72,64.22, 47.06, 46.35, 42.77, 39.58, 37.81, 37.64, 37.55, 36.75, 35.48,35.02, 32.59, 32.52, 30.27, 28.43, 27.56, 27.52, 26.92, 24.62, 23.34,23.25, 23.10, 18.24, 12.64; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 1025.4207 (100%), calcd. 1025.4189.

Compound 38: The suspension of 37 (0.227 g, 0.227 mmol) and NaN₃ (0.147g, 2.27 mmol) in dry DMSO (5 mL) was stirred at 80° C. overnight,diluted with H₂ O (50 mL) and extracted with EtOAc (3×20 mL). Theextracts were washed with brine once and dried over anhydrous Na₂ SO₄.SiO₂ chromatography (EtOAc/hexanes 1:8) afforded the desired product(0.153 g, 80% yield) as a yellow oil. IR (neat) 2929, 2866, 2105, 1490,1466, 1448, 1107, 705 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6H), 7.32-7.19 (m, 9 H), 3.80-3.74 (m, 1 H), 3.70-3.55 (series ofmultiplets, 5 H), 3.41-3.19 (series of multiplets, 9 H), 3.04-2.98 (m, 2H), 2.41 (td, J=13.1, 2.44 Hz, 1 H), 2.29-2.14 (m, 2 H), 2.04-0.86(series of multiplets, 20 H), 0.93 (s, 3 H), 0.91 (d, J=6.60 Hz, 3 H),0.66 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.78, 128.93, 127.87, 126.96,86.46, 81.30, 77.16, 75.21, 67.99, 67.44, 67.03, 64.41, 51.64, 51.57,51,33, 46.71, 46.30, 42.35, 39.75, 36.72, 35.64, 35.20, 32.52, 32.42,30.17, 28.63, 27.80, 27.22, 26.90, 24.80, 23.55, 23.30, 23.24, 18.23,12.65; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 866.5049(96.9%), calcd. 866.5057.

Compound 39: p-Toluenesulfonic acid (1.72 mg) was added into thesolution of 38 (0.153 g, 0.18 mmol) in CH₂ Cl₂ (5 mL) and MeOH (5 mL),and the mixture was stirred for 2.5 hours. Saturated NaHCO₃ solution (5mL) was introduced followed by the introduction of brine (30 mL). Theaqueous mixture was extracted with EtOAc and the combined extractswashed with brine and dried over Na₂ SO₄. The desired product (0.10 g,92% yield) was obtained as a pale yellowish oil after SiO₂chromatography (EtOAc/hexanes 1:3). IR (neat) 3426, 2926, 2104, 1467,1441, 1347, 1107 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.81-3.74 (m, 1 H),3.71-3.54 (m, 7 H), 3.41-3.19 (m, 9 H), 2.41 (td, J=13.61, 2.32 Hz, 1H), 2.30-2.14 (m, 2 H), 2.07-1.98 (m, 1H), 1.94-0.95 (series ofmultiplets, 21 H), 0.95 (d, J=6.35 Hz, 3 H), 0.93 (s, 3 H), 0.69 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 81.22, 77.08, 75.13, 67.94, 67.36, 66.97,63.76, 51.59, 51.51, 51.26, 46.51, 46.24, 42.31, 39.68, 36.64, 35.58,35.12, 32.34, 31.92, 30.11, 29.55, 28.54, 27.82, 27.16, 24.75, 23.47,23.23, 23.18, 18.15, 12.56; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 624.3966 (54.9%), calcd. 624.3962.

Compound 40: To a solution of 39 (0.10 g, 0.166 mmol) in CH₂Cl₂ (8 mL)at 0° C. was added NEt₃ (34.8 μL, 0.25 mmol) under N₂ followed by theintroduction of mesyl chloride (15.5 .mu.L, 0.199 mmol). The mixture wasstirred 15 minutes. Addition of H₂ O (3 mL) and brine (20 mL) wasfollowed by extraction with EtOAc (4×10 mL). The combined extracts werewashed with brine once and dried over Na₂ SO₄. After removal of solvent,the residue was mixed with N-benzylmethylamine (0.5 mL) and heated to80° C. under N₂ overnight. Excess N-benzyl methylamine was removed invacuo and the residue was subjected to SiO₂ chromatography(EtOAc/hexanes 1:4) to give the product (0.109 g, 93% yield) as a yellowoil. IR (neat) 2936, 2784, 2103, 1467, 1442, 1346, 1302, 1106, 1027cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.23 (m, 5 H), 3.81-3.74 (m, 1 H),3.71-3.55 (m, 5 H), 3.47 (s, 2 H), 3.41-3.19 (m, 9 H), 2,46-2.11 (m, 5H), 2.18 (s, 3 H), 2.03-0.85 (series of multiplets, 20 H), 0.93 (s, 3H), 0.93 (d, J=6.35 Hz, 3 H,), 0.67 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ139.54, 129.26, 128.32, 126.97, 81.26, 77.12, 75.17, 67.98, 67.42,67.00, 62.50, 58.41, 51.61, 51.54, 51.29, 46.66, 46.28, 42.46, 42.32,39.72, 36.68, 35.76, 35.16, 33.75, 32.38, 30.15, 28.59, 27.85, 27.19,24.77, 24.15, 23.53, 23.28, 23.22, 18.28, 12.60; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 705.4929 (100%), calcd.705.4928.

Compound 8: A suspension of 40 (0.109 g, 0.155 mmol) and LiAlH₄ (23.5mg, 0.62 mmol) in THF (20 mL) was stirred under N₂ overnight. Na₂SO₄.10H₂ O was carefully added and stirred until no grey colorpersisted. Anhydrous Na₂SO₄ was added and the white precipitate wasfiltered out and rinsed with dry THF. After removal of solvent, theresidue was dissolved in minimum CH₂Cl₂ and filtered. The desiredproduct (0.091 g, 94% yield) was obtained as a colorless oil after thesolvent was removed. IR (neat) 3371, 3290, 3027, 2938, 2862, 2785, 1586,1493, 1453, 1377, 1347, 1098 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.31-7.21(m, 5 H), 3.65-3.53 (m, 4 H), 3.47 (s, 2 H), 3.42-3.34 (m, 2 H), 3.30(bs, 1 H), 3.26-3.20 (m, 1H), 3.14-3.09 (m, 1 H), 2.89-2.81 (m, 6 H),2.39-2.27 (m, 3 H), 2.17 (s, 3 H), 2.15-0.88 (series of multiplets, 29H), 0.93 (d, J=6.59 Hz, 3 H), 0.92 (s, 3 H), 0.67 (s, 3 H); ¹³C NMR(CDCl₃, 75 MHz) δ 139.34, 129.16, 128.24, 126.90, 80.75, 76.44, 74.29,70.58, 69.88, 69.75, 62.47, 58.27, 46.66, 46.47, 42.75, 42.63, 42.51,42.35, 39.77, 36.87, 35.73, 35.04, 33.77, 32.90, 30.38, 28.71, 27.70,27.32, 24.89, 24.09, 23.53, 23.36, 23.25, 18.24, 12.62; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 627.5199 (23.3%), calcd.627.5213.

Compound CSA-7: To a solution of 23 (0.18 g, 0.28 mmol) in dry DMF (4mL) were added NaH (0.224 g, 60% in mineral oil, 5.60 mmol) and 1-bromooctane (0.48 mL, 2.80 mmol). The suspension was stirred under N₂ at 65°C. overnight followed by the introduction of H₂ O (60 mL) and extractionwith ether (4×20 mL). The combined extracts were washed with brine anddried over Na₂ SO₄. SiO₂ chromatography (hexanes and 5% EtOAc inhexanes) afforded the desired product (0.169 g, 80% yield) as a paleyellowish oil. IR (neat) 2927, 2865, 2099, 1478, 1462, 1451, 1350, 1264,1105 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.69-3.35 (series of multiplets, 15H), 3.26-3.02 (series of multiplets, 4 H), 2.19-2.02 (m, 3 H), 1.97-1.16(series of multiplets, 37 H), 1.12-0.99 (m, 2 H), 0.92-0.86 (m, 9 H),0.65 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.69, 79.84, 76.13, 71.57,71.15, 65.07, 64.49, 64.39, 49.08, 48.99, 48.80, 46.68, 46.45, 42.72,42.05, 39.88, 35.74, 35.49, 35.36, 35.14, 32.42, 32.03, 30.01, 29.85,29.81, 29.76, 29.67, 29.48, 29.14, 27.92, 27.80, 27.70, 26.58, 26.42,23.59, 23.09, 22.92, 22.86, 18.11, 14.31, 12.65; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 778.5685 (22.1%), calcd.778.5683. The triazide (0.169 g, 0.224 mmol) and LiAlH₄ (0.025 g, 0.67mmol) were suspended in anhydrous THF (10 mL) and stirred under N₂ atroom temperature overnight followed by careful introduction of Na₂ SO₄hydrate. After the grey color disappeared, anhydrous Na₂ SO₄ was addedand stirred. The white precipitate was removed by filtration and washedwith THF. After removal of solvent, the residue was dissolved in 1 Mhydrochloric acid and the aqueous solution was extracted with ether (5mL) once. The aqueous solution was then made basic by adding 20% aqueousNaOH solution followed by extraction with Et₂ O (4×5 mL). The combinedextracts were washed, dried and concentrated. The residue was thensubject to SiO₂ chromatography (MeOH/CH₂Cl₂ (1:1) followed byMeOH/CH₂Cl₂/NH₃. H₂ O (4:4:1)) to afford the desired product (0.091 g,60% yield) as a colorless oil. IR (neat) 3361, 2927, 2855, 1576, 1465,1351, 1105 cm⁻¹; ¹H NMR (CD₃OD, 300 MHz) δ 4.86 (bs, 6 H), 3.77-3.72 (m,1 H), 3.70-3.61 (m,1 H), 3.57-3.53 (m, 3 H), 3.43-3.38 (m, 4 H),3.34-3.27 (m, 2 H), 3.18-3.10 (m, 2 H), 2.84-2.71 (m, 6 H), 2.22-2.07(m, 3 H), 2.00-1.02 (series of multiplets, 39 H), 0.97-0.88 (m, 9 H),0.71 (s, 3 H); ¹³C NMR (CD₃ OD, 75 MHz) δ 82.20, 81.00, 77.62, 72.52,72.06, 68.00, 67.92, 67.39, 48.20, 47.53, 44.26, 43.40, 41.42, 41.15,40.84, 40.35, 36.88, 36.73, 36.42, 36.11, 34.24, 34.05, 33.94, 33.67,33.17, 30.95, 30.72, 30.62, 29.81, 29.35, 28.87, 28.79, 27.51, 24.57,23.90, 23.83, 23.44, 18.76, 14.62, 13.07; HRFAB-MS (thioglycerol matrix)m/e: ([M+H]⁺) 678.6133 (100%), calcd. 678.6149.

Compound CSA-8: A suspension of 23 (0.126 g, 0.196 mmol) and LiAlH₄(0.037 g, 0.98 mmol) in THF (40 mL) was stirred at room temperatureunder N₂ overnight followed by careful addition of Na₂SO₄.10H₂O. Afterthe grey color in the suspension disappeared, anhydrous Na₂SO₄ was addedand stirred until organic layer became clear. The white precipitate wasremoved by filtration and washed with twice THF. The THF was removed invacuo, and the residue was subject to SiO₂ chromatography(MeOH/CH₂Cl₂/NH₃/H₂O (4:4:1)) to afford the desired product (0.066 g,60% yield) as a colorless oil. IR (neat) 3365, 2933, 2865, 1651, 1471,1455, 1339, 1103 cm⁻¹; ¹H NMR (CDCl₃/30% CD₃OD, 300 MHz) δ 4.43 (bs, 7H), 3.74-3.68 (m, 1 H), 3.66-3.60 (m, 1 H), 3.57-3.50 (m, 5 H),3.34-3.25 (M, 2 H), 3.17-3.06 (M, 2 H), 2.84-2.74 (M, 6 H), 2.19-2.01(M, 3 H), 1.97-0.96 (series of multiplets, 27 H), 0.94 (d, J=7.2 Hz, 3H), 0.92 (s, 3 H), 0.69 (s, 3 H); ¹³ C NMR (CDCl₃, 75 MHz) δ 80.44,79.27, 75.77, 66.59, 66.53, 65.86, 62.51, 46.21, 45.84, 42.55, 41.53,40.09, 39.43, 39.31, 39.02, 35.16, 34.93, 34.86, 34.57, 32.93, 32.71,31.57, 28.66, 28.33, 27.64, 27.22, 23.04, 22.40, 22.29, 17.60, 11.98;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 566.4889 (8.9%), calcd.566.4897.

Example 5

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds CSA-11 and 43-47.

Compound 43: Precursor compound 41 was prepared following the methodreported by D. H. R. Barton, J. Wozniak, S. Z. Zard, Tetrahedron, 1989,vol. 45, 3741-3754. A mixture of 41 (1.00 g, 2.10 mmol), ethylene glycol(3.52 mL, 63 mmol) and p-TsOH (20 mg, 0.105 mmol) was refluxed inbenzene under N₂ for 16 hours. Water formed during the reaction wasremoved by a Dean-Stark moisture trap. The cooled mixture was washedwith NaHCO₃ solution (50 mL) and extracted with Et₂O (50 mL, 2×30 mL).The combined extracts were washed with brine and dried over anhydrousNa₂ SO₄. Removal of the solvent gave the product (1.09 g, 100%) as awhite glass. IR (neat) 2939, 2876, 1735, 1447, 1377, 1247, 1074, 1057,1039 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 5.10 (t, J=2.70 Hz, 1 H), 4.92 (d,J=2.69 Hz, 1 H), 4.63-4.52 (m, 1 H), 3.98-3.80 (m, 4 H), 2.323 (t,J=9.51 Hz, 1 H), 2.13 (s, 3 H), 2.08 (s, 3 H), 2.05 (s, 3 H), 2.00-1.40(series of multiplets, 15 H), 1.34-0.98 (m, 3 H), 1.20 (s, 3 H), 0.92(s, 3 H), 0.82 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 170.69, 170.63,170.47, 111.38, 75.07, 74.23, 70.85, 64.95, 63.43, 49.85, 44.73, 43.39,41.11, 37.37, 34.84, 34.80, 34.52, 31.42, 29.18, 27.02, 25.41, 24.16,22.72, 22.57, 22.44, 21.73, 21.63, 13.40; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+H]⁺) 521.3106 (38.6%), calcd. 521.3114. The triacetate(1.09 g, 2.10 mmol) was dissolved in MeOH (50 mL). NaOH (0.84 g, 21mmol) was added to the solution. The suspension was then refluxed underN₂ for 24 hours. MeOH was then removed in vacuo and the residue wasdissolved in Et₂ O (100 mL) and washed with H₂ 0, brine, and then driedover anhydrous Na₂ SO₄. The desired product (0.80 g, 96% yield) wasobtained as white solid after removal of solvent. m.p. 199-200° C. IR(neat) 3396, 2932, 1462, 1446, 1371, 1265, 1078, 1055 cm⁻¹; ¹H NMR (10%CD₃ OD in CDCl₃, 300 MHz) δ 4.08-3.83 (series of multiplets, 9 H),3.44-3.34 (m, 1 H), 2.41 (t, J=9.28 Hz, 1 H), 2.22-2.10 (m, 2 H),1.96-1.50 (series of multiplets, 12 H), 1.45-0.96 (series of multiplets,4 H), 1.32 (s, 3 H), 0.89 (s, 3 H), 0.78 (s, 3 H); ¹³C NMR (10% CD₃OD inCDCl₃, 75 MHz) δ 112.11, 72.35, 71.57, 68.09, 64.54, 63.24, 49.36,45.90, 41.48, 41.45, 39.18, 38.79, 35.29, 34.71, 34.45, 29.90, 27.26,26.60, 23.65, 22.54, 22.44, 22.35, 13.46; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 417.2622 (87.3%), calcd. 417.2617.

Compound 44: To a round-bottom flask were added 43 (0.80 g, 2.03 mmol)and dry THF (100 mL) followed by the addition of NaH (60% in mineraloil, 0.81 g, 20.3 mmol). The suspension was refluxed under N₂ for 30minutes before the addition of allyl bromide (1.75 mL, 20.3 mmol). After48 hours of reflux, another 10 eq. of NaH and allyl bromide were added.After another 48 hours, TLC showed no intermediates left. Cold water (50mL) was added to the cooled suspension. The resulted mixture wasextracted with Et₂O (60 mL, 2×30 mL). The combined extracts were washedwith brine and dried over anhydrous Na₂SO₄. SiO₂ column chromatography(6% EtOAc in hexanes) gave the desired product (0.94 g, 90% yield) as apale yellow oil. IR (neat) 3076, 2933, 2866, 1645, 1446, 1423, 1408,1368, 1289, 1252, 1226, 1206, 1130, 1080, 1057cm⁻¹; ¹H NMR (CDCl₃, 300MHz) δ 6.02-5.84 (m, 3 H), 5.31-5.04 (m, 6 H), 4.12-4.05 (m, 2 H),4.01-3.81 (m, 7 H), 3.70 (dd, J=12.94, 5.62 Hz, 1 H), 3.55 (t, J=2.56Hz, 1 H), 3.33 (d, J=2.93 Hz, 1 H), 3.18-3.08 (m, 1 H), 2.65 (t, J=10.01Hz, 1 H), 2.32-2.14 (m, 3 H), 1.84-1.45 (series of multiplets, 10 H),1.41-1.22 (m, 3 H), 1.27 (s, 3 H), 1.14-0.92 (m, 2 H), 0.89 (s, 3 H),0.75 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 136.38, 136.07, 136.00, 116.31,115.54, 115.38, 112.34, 80.07, 79.22, 75.05, 69.83, 69.34, 68.82, 65.14,63.24, 48.80, 45.96, 42.47, 42.15, 39.40, 35.55, 35.16, 35.15, 29.04,28.22, 27.52, 24.21, 23.38, 23.11, 22.95, 22.58, 13.79; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 537.3549 (100%), calcd.537.3556.

Compound 45: To the solution of 44 (0.94 g, 1.83 mmol) in dry THF (50mL) was added 9-BBN (0.5 M solution in THF, 14.7 mL, 7.34 mmol) and themixture was stirred under N₂ at room temperature for 12 hours before theaddition of 20% NaOH solution (4 mL) and 30% H₂ O₂ solution (4 mL). Theresulted mixture was then refluxed for an hour followed by the additionof brine (100 mL) and extracted with EtOAc (4×30 mL). The combinedextracts were dried over anhydrous Na₂SO₄. After the removal of solvent,the residue was purified by SiO₂ column chromatography (EtOAc followedby 10% MeOH in CH₂Cl₂) to give the product (0.559 g, 54% yield) as acolorless oil. IR (neat) 3410, 2933, 2872, 1471, 1446, 1367, 1252, 1086cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 4.02-3.52 (series of multiplets, 17 H),3.41-3.35 (m, 1 H), 3.29 (d, J=2.44 Hz, 1 H), 3.22-3.15 (m, 3 H), 2.58(t, J=10.01 Hz, 1 H), 2.27-1.95 (m, 3 H), 1.83-1.48 (series ofmultiples, 16 H), 1.40-0.93 (series of multiplets, 5 H), 1.27 (s, 3 H),0.90 (s, 3 H), 0.75 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 112.41, 80.09,79.09, 76.31, 66.70, 66.02, 65.93, 64.80, 63.26, 61.53, 61.25, 60.86,48.59, 45.80, 42.51, 41.72, 39.10, 35.36, 35.02, 34.98, 32.87, 32.52,32.40, 28.88, 27.94, 27.21, 24.33, 23.02, 22.84 (2 C's), 22.44, 13.69;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 591.3881 (100%),calcd. 591.3873.

Compound 46: To a solution of 45 (0.559 g, 0.98 mmol) in acetone (40 mL)and water (4 mL) was added PPTS (0.124 g, 0.49 mmol) and the solutionwas refluxed under N₂ for 16 hours. The solvent was removed underreduced pressure. Water (40 mL) was then added to the residue and themixture was extracted with EtOAc (40 mL, 2×20 mL). The combined extractswere washed with brine, dried and evaporated to dryness. SiO₂ columnchromatography (8% MeOH in CH₂Cl₂) of the residue afforded the desiredproduct (0.509 g, 98% yield) as clear oil. IR (neat) 3382, 2941, 2876,1699, 1449, 1366, 1099 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.83-3.72 (m, 8H), 3.66 (t, J=5.62 Hz, 2 H), 3.54 (bs, 2 H), 3.43-3.28 (m, 4 H),3.24-3.12 (m, 2 H), 2.26-2.00 (m, 4 H), 2.08 (s, 3 H), 1.98-1.50 (seriesof multiplets, 15 H), 1.42-0.96 (series of multiplets, 6 H), 0.90 (s, 3H), 0.62 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 210.49, 78.87 (2 C's),76.30, 66.86, 66.18, 65.69, 61.74, 61.43, 60.71, 55.31, 48.05, 43.02,41.58, 39.53, 35.28, 35.09, 34.96, 32.77, 32.70, 32.31, 31.12, 28.72,27.88, 27.14, 23.47, 22.75, 22.47, 22.34, 13.86; HRFAB-MS(thioglycerol+Na⁺ matrix) m/c: ([M+Na]⁺) 547.3624 (100%), calcd.547.3611.

Compound 47: To a solution of 46 (0.18 g, 0.344 mmol) in dry CH₂Cl₂ (10mL) at 0° C. was added Et₃ N (0.168 mL, 1.20 mmol) followed by theaddition of mesyl chloride (0.088 mL, 1.13 mmol). After 10 minutes, H₂O(3 mL) and brine (30 mL) were added. The mixture was extracted withEtOAc (30 mL, 2×10 mL) and the extracts were washed with brine and driedover anhydrous Na₂ SO₄. After removal of solvent, the residue wasdissolved in DMSO (5 mL) and NaN₃ (0.233 g, 3.44 mmol). The suspensionwas heated up to 50° C. under N₂ for 12 hours. H₂ O (50 mL) was added tothe cool suspension and the mixture was extracted with EtOAc (30 mL,2×10 mL) and the extracts were washed with brine and dried overanhydrous Na₂ SO₄. SiO₂ column chromatography (EtOAc/hexanes 1:5)afforded the product (0.191 g, 88% yield for two steps) as a pale yellowoil. IR (neat) 2933, 2872, 2096, 1702, 1451, 1363, 1263, 1102 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ 3.72-3.64 (m, 2 H), 3.55-3.24 (series ofmultiplets, 1I1 H), 3.18-3.02 (m, 2 H), 2.22-2.02 (m, 4 H), 2.08 (s, 3H), 1.95-1.46 (series of multiplets, 15 H), 1.28-0.96 (series ofmultiplets, 6 H), 0.89 (s, 3 H), 0.62 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz)δ 210.36, 79.69, 79.22, 75.98, 65.08, 64.80, 64.53, 55.31, 48.93, 48.86,48.76, 48.06, 43.03, 41.91, 39.66, 35.44, 35.31, 35.12, 31.04, 29.77,29.69, 29.67, 28.99, 28.10, 27.65, 23.60, 22.99, 22.95, 22.50, 14.00;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 622.3820 (100%),calcd. 622.3805.

Compound CSA-11: Compound 47 (0.191 g, 0.319 mmol) was dissolved in dryTHF (20 mL) followed by the addition of LiAlH₄ (60.4 mg, 1.59 mmol). Thegrey suspension was stirred under N₂ at room temperature for 12 hours.Na₂SO₄.10H₂O powder was carefully added. After the grey color in thesuspension disappeared, anhydrous Na₂SO₄ was added and the precipitatewas filtered out. After the removal of solvent, the residue was purifiedby column chromatography (silica gel, MeOH/CH₂Cl₂/28% NH₃.H₂ O 3:3:1).After most of the solvent was rotavapped off from the fractionscollected, 5% HCl solution (2 mL) was added to dissolve the milkyresidue. The resulted clear solution was then extracted with Et₂O (2×10mL). 20% NaOH solution was then added until the solution became stronglybasic. CH₂Cl₂ (20 mL, 2×10 mL) was used to extract the basic solution.The combined extracts were dried over anhydrous Na₂SO₄ and removal ofsolvent gave the desired product (0.115 g, 69% yield) as a colorlessoil. From ¹H NMR it appears that this compound was a mixture of twostereoisomers at C₂₀ with a ratio of approximately 9:1. Thestereoisomers were not separated, but used as recovered. Spectra for themost abundant isomer: IR (neat) 3353, 2926, 2858, 1574, 1470, 1366, 1102cm⁻¹; ¹H NMR (20% CDCl₃ in CD₃ OD, 300 MHz) δ 4.69 (bs, 7 H), 3.76-3.69(m, 1 H), 3.63-3.53 (m, 5 H), 3.50-3.40 (m, 1 H), 3.29 (bs, 1 H),3.18-3.07 (m, 2 H), 2.94-2.83 (m, 1 H), 2.81-2.66 (m, 5 H), 2.23-2.06(m, 4 H), 1.87-1.50 (series of multiplets, 15 H), 1.39-0.96 (series ofmultiplets, 6 H), 1.11 (d, J−6.10 Hz, 3 H), 0.93 (s, 3 H), 0.75 (s, 3H); ¹³C NMR (20% CDCl₃ in CD₃ OD, 75 MHz) δ 81.46, 80.67, 77.32, 70.68,67.90, 67.66, 67.18, 50.32, 47.17, 43.30, 43.06, 40.74, 40.64, 40.38,40.26, 36.31, 36.28, 35.93, 34.30, 34.02, 33.29, 29.63, 29.31, 28.43,26.10, 24.67, 24.09, 23.96, 23.50, 13.30 for the major isomer; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 524.4431 (64.2%), calcd.524.4427.

Example 6

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds CSA-10 and 48-49

Compound 48: To a solution of 23 (0.15 g, 0.233 mmol) in dry CH₂ Cl₂ (15mL) at 0° C. was added Et₃ N (48.8 μL, 0.35 mmol) followed by theaddition of CH₃SO₂Cl (21.7 μL, 0.28 mmol). The mixture was stirred for15 minutes before H₂O (3 mL) was added. Saturated NaCl solution (20 mL)was then added, and the mixture was extracted with EtOAc (40 mL, 2×20mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The solvent was rotovapped off and to the residue wereadded NaBr (0.12 g, 1.17 mmol) and DMF (10 mL). The suspension washeated up to 80° C. under N₂ for 2 hours. DMF was removed under vacuumand the residue was chromatographed on silica (EtOAc/hexanes 1:10) togive the desired product (0.191 g, 97% yield) as a pale yellow oil. ¹ HNMR (CDCl₃, 300 MHz) δ 3.69-3.35 (series of multiplets, 13 H), 3.28-3.02(series of multiplets, 4 H), 2,18-2.04 (m, 3 H), 2.00-1.60 (series ofmultiplets, 16 H), 1.58-0.96 (series of multiplets, 11 H), 0.92 (d,J=6.34 Hz, 3 H), 0.89 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ80.62, 79.81, 76.08, 65.07, 64.50, 64.34, 49.03, 48.98, 48.79, 46.49,46.46, 42.73, 42.02, 39.85, 35.47, 35.34, 35.12, 34.79, 34.72, 29.82,29.80, 29.74, 29.11, 27.91, 27.78, 27.69, 23.55, 23.07, 22.88, 18.10,12.62; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M−H]⁺) 706.3609(63.1%), calcd. 706.3591; 704.3616 (52.8%), calcd. 704.3611.

Compound 49: Compound 48 (0.191 g, 0.269 mmol) and 23 (0.295 g, 0.459mmol) was dissolved in DMF (3 mL, distilled over BaO at 6 mm Hg beforeuse) followed by the addition of NaH (0.054 g, 60% in mineral oil). Thesuspension was stirred under N₂ at room temperature for 24 hours. H₂ O(100 mL) was added to quench excess NaH and the mixture was thenextracted with Et₂ O (40 mL, 3×20 mL) and the combined extracts werewashed with brine and dried over anhydrous Na₂ SO₄. The desired product(0.177 g, 52% yield based on compound 23) was obtained as a pale yellowoil after SiO₂ chromatography (EtOAc/hexanes 1:6, then 1:2). IR (neat)2940, 2862, 2095, 1472, 1456, 1362, 1263, 1113 cm⁻¹; ¹H NMR(CDCl₃, 300MHz) δ 3.68-3.35 (series of multiplets, 26 H), 3.28-3.02 (series ofmultiplets, 8 H), 2.20-2.04 (m, 6 H), 1.96-1.60 (series of multiplets,30 H), 1.52-0.98 (series of multiplets, 12 H), 0.91 (d, J=6.59 Hz, 6 H),0.89 (s, 6 H), 0.65 (s, 6 H); ¹³C NMR(CDCl₃, 75 MHz) δ 80.68, 79.83,76.13, 71.71, 65.06, 64.48, 64.39, 49.08, 48.98, 48.80, 46.64, 46.44,42.71, 42.04, 39.88, 35.73, 35.49, 35.36, 35.14, 32.41, 29.84, 29.81,29.76, 29.14, 27.92, 27.78, 27.69, 26.58, 23.59, 23.08, 22.92, 18.12,12.64.

Compound CSA-10: Compound 49 (0.219 g, 0.173 mmol) was dissolved in dryTHF (10 mL) followed by the addition of LiAlH₄ (65 mg, 1.73 mmol). Thegrey suspension was stirred under N₂ at room temperature for 12 hours.Na₂SO₄.10H₂O powder was carefully added. After the grey color in thesuspension disappeared, anhydrous Na₂SO₄ was added and the precipitatewas filtered out. After the removal of solvent, the residue was purifiedby column chromatography (silica gel, MeOH/CH₂Cl₂/28% NH₃.H₂O2.5:2.5:1). After most of the solvent was rotavapped off from thefractions collected, 5% HCl solution (2 mL) was added to dissolve themilky residue. The resulted clear solution was then extracted with Et₂O(2×10 mL). 20% NaOH solution was then added until the solution becamestrongly basic. CH₂Cl₂ (20 mL, 2×10 mL) was used to extract the basicsolution. The combined extracts were dried over anhydrous Na₂SO₄ andremoval of solvent gave the desired product (0.147 g, 76% yield) as awhite glass. IR (neat) 3364, 3287, 2934, 2861, 1596, 1464, 1363, 1105cm⁻¹; ¹H NMR (20% CDCl₃ in CD₃OD, 500 MHz) δ 4.74 (bs, 12 H), 3.75-3.70(m, 2 H), 3.65-3.61 (m, 2 H), 3.57-3.52 (m, 6 H), 3.40 (t, J=3.60 Hz, 4H), 3.30 (bs, 4 H), 3.16-3.10 (m, 4 H), 2.84-2.73 (m, 12 H), 2.18-2.07(m, 6 H), 1.97-1.61 (series of multiplets, 30 H), 1.58-0.98 (series ofmultiplets, 24 H), 0.95 (d, J=6.84 Hz, 6 H), 0.94 (s, 6 H), 0.70 (s, 6H); ¹³C NMR (20% CDCl₃ in CD₃OD, 125 MHz) δ 81.70, 80.52, 77.09, 72.34,67.75 (2 C's), 67.07, 47.80, 47.13, 43.76, 42.87, 41.20, 40.65, 40.58,40.14, 36.43, 36.25, 36.08, 35.77, 34.15, 33.87 (2 C's), 33.18, 29.55,28.92, 28.47, 28.42, 27.25, 24.27, 23.54, 23.41, 18.70, 13.07; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 1113.9625 (68.8%), calcd.1113.9610.

Example 7

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 111-113 and 116a-d.

Compounds 116a-d: Representative procedure: preparation of 116b. NaH(0.06 g, 60% in mineral oil, 1.49 mmol) and propyl bromide (0.136 mL,1.49 mmol) were added to a DMF solution of compound 23 (described in Liet al., J. Am. Chem. Soc. 1998, 120, 2961) (0.096 g, 0.149 mmol). Thesuspension was stirred under N₂ for 24 hr. H₂O (20 mL) was added, andthe mixture was extracted with hexanes (3×10 mL). The combined extractswere dried over Na₂SO₄ and concentrated in vacuo. Silica gelchromatography (10% EtOAc in hexanes) afforded the desired product (92mg, 90% yield) as a pale yellow oil. ¹H NMR (CDCl₃, 500 MHz) δ 3.68-3.64(m, 1 H), 3.61-3.57 (m, 1 H), 3.52 (t, J=6.1 Hz, 2 H), 3.49 (bs, 1 H),3.46-3.35 (m, 10 H), 3.25 (d, J=2.4 Hz, 1 H), 3.23-3.19 (m, 1 H),3.16-3.11 (m, 1 H), 3.09-3.03 (m, 1 H), 2.17-2.03 (m, 3 H), 1.95-1.55(m, 17 H), 1.51-1.40 (m, 4 H), 1.38-1.17 (m, 5 H), 1.11-0.96 (m, 3 H),0.93-0.89 (m, 9 H), 0.65 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.64,79.79, 76.08, 72.67, 71.59, 65.01, 64.44, 64.33, 49.04, 48.94, 48.75,46.61, 46.40, 42.68, 42.00, 39.83, 35.72, 35.45, 35.30, 35.10, 32.38,29.81, 29.77, 29.72, 29.09, 27.88, 27.76, 27.65, 26.52, 23.55, 23.12,23.04, 22.87, 18.06, 12.60, 10.79; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+Na]⁺) 708.4910 (23.5%), calcd. 708.4920.

Compounds 111, CSA-17, and 113: Representative procedure: preparation ofCSA-17.

Compound 116b (0.092 g, 0.134 mmol) was dissolved in THF (10 mL)followed by the addition of LiAlH₄ (0.031 g, 0.81 mmol). The suspensionwas stirred under N₂ for 12 hr. Na₂SO₄.10H₂ O (about 1 g) was thencarefully added. After the gray color in the suspension dissipated,anhydrous Na₂SO₄ was added, and the precipitate was removed byfiltration. Concentration and silica gel chromatography (CH₂Cl₂/MeOH/28%NH₃.H₂O 12: 6:1, then 10:5:1) yielded a glass which was dissolved in 1 MHCl (2 mL). The resulting clear solution was washed with Et₂O (2×10ml.). 20% NaOH solution was added to the aqueous phase until thesolution became strongly basic. CH₂Cl₂ (3×10 mL) was used to extract thebasic solution. The combined extracts were dried over anhydrous Na₂SO₄and concentrated in vacuo to give the desired product (0.045 g, 55%yield) as a white glass. ¹H NMR (about 20% CDCl₃ in CD₃OD, 500 MHz) δ4.73 (bs, 6 H), 3.74-3.70 (m, 1 H), 3.65-3.61 (m, 1 H), 3.55 (t, J=6.3Hz, 2 H), 3.42-3.38 (m, 4 H), 3.33-3.30 (m, 2 H), 3.16-3.10 (m, 2 H),2.83-2.73 (m, 6 H), 2.18-2.06 (m, 3 H), 1.96-1.20 (series of multiplets,26 H), 1.12-0.98 (m, 3 H), 0.95-0.92 (m, 9 H), 0.70 (s, 3 H); ¹³C NMR(about 20% CDCl₃ in CD₃OD, 75 MHz) δ 81.67, 80.49, 77.04, 73.44, 72.28,67.77, 67.71, 67.06, 47.74, 47.08, 43.75, 42.82, 41.21, 40.60, 40.56,40.12, 36.47, 36.19, 36.04, 35.74, 34.09, 33.82, 33.78, 33.16, 29.49,28.87, 28.43, 27.18, 24.22, 23.66, 23.49, 23.40, 18.64, 13.04, 11.03;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 608.5348 (100%), calcd.608.5330. 111: ¹H NMR (about 20% CDCl₃ in CD₃OD, 500 MHz) δ 4.79 (bs,6H), 3.74-3.71 (m, 1 H), 3.66-3.62 (m, 1 H), 3.55 (t, J=6.1 Hz, 2 II),3.52 (bs, 1 II), 3.38-3.28 (series of multiplets, 4 H), 3.33 (s, 3 H),3.16-3.10 (m, 2H), 2.83-2.72 (m, 6 H), 2.19-2.07 (m, 3 H), 1.97-1.62(series of multiplets, 15 H), 1.58-1.20 (series of multiplets, 9 H),1.13-0.98 (m, 3 H), 0.95 (d, J=6.3 Hz, 3 H), 0.93 (s, 3 H), 0.70 (s, 3H); ¹³C NMR (about 20% CDCl₃ in CD₃OD, 75 MHz) δ 81.82, 80.65, 77.20,74.43, 67.85, 67.18, 58.90, 47.80, 47.22, 43,91, 43.01, 41.31, 40.78,40.69, 40.22, 36.63, 36.35, 36.18, 35.86, 34.27, 33.97, 33.26, 29.60,29.03, 28.58, 28.53, 27.14, 24.33, 23.61, 23.45, 18.68, 13.06; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 602.4855 (100%), calcd.602.4873. 113: ¹H NMR (about 50% CDCl₃ in CD₃OD, 500 MHz) δ 4.08 (bs, 6H), 3.71-3.67 (m, 1 H), 3.62-3.58 (m, 1 H), 3.53 (t, J=6.3 Hz, 2 H),3.49 (bs, 1 H), 3.43-3.38 (m, 4 H), 3.31-3.27 (m, 2 H), 3.14-3.07 (m, 2H), 2.83-2.73 (m, 6 H), 2.16-2.03 (m, 3 H), 1.93-1.17 (series ofmultiplets, 30 H), 1.10-0.96 (m, 3 H), 0.93-0.89 (m, 9 H), 0.67 (s, 3H); ¹³C NMR (about 50% CDCl₃ in CD₃OD, 75 MHz) δ 80.51, 79.35, 75.85,71.29, 70.83, 66.73, 66.62, 65.96, 46.68, 45.98, 42.59, 41.63, 40.20,39.53, 39.43, 39.21, 35.34, 35.04, 35.00, 34.71, 33.11, 32.90, 32.82,32.00, 29.15, 28.49, 28.15, 27.75, 27.35, 26.22, 23.18, 22.60, 22.45,22.34, 17.77, 13.75, 12.22; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+H]⁺) 636.5679 (100%), calcd. 636.5669.

Example 8

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 106 and 124.

Compound 124: Compound 47 (0.256 g, 0.489 mmol) was dissolved in CH₂Cl₂(10 mL), and cooled to 0° C. followed by the addition of Na₂HPO₄ (0.69g, 4.89 mmol) and urea-hydrogen peroxide complex (UHP) (0.069 g, 0.733mmol). Trifluoroacetic anhydride (TFAA) (0.138 mL, 0.977 mmol) was thenadded dropwise. The suspension was stirred for 12 hr, and additional UHP(23 mg, 0.25 mmol) and TFAA (0.069 mL, 0.49 mmol) were added. Afteranother 12 hr, H₂O (30 mL) was added, and the resulting mixture wasextracted with EtOAc (3×20 mL). The combined extracts were washed withbrine (50 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo.SiO₂ chromatography (EtOAc/hexanes 1:5) afforded the desired product(0.145 g, 55% yield) as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 5.21(dd, J=9.3 and 7.3 Hz, 1 H), 3.70-3.57 (m, 2 H), 3.55 (t, J=6.0 Hz, 2H), 3.43-3.37 (m, 6 H), 3.32-3.25 (m, 3 H), 3.17-3.02 (m, 2 H),2.28-2.05 (m, 4 H), 2.03 (s, 3 H), 1.86-1.19 (series of multiplets, 19H), 0.97 (dd, J=14.5 and 3.3 Hz, 1 H), 0.90 (s, 3 H), 0.78 (s, 3 H); ³CNMR (CDCl₃, 75 MHz) δ 171.08, 79.71, 78.03, 75.72, 75.53, 65.41, 65.04,64.53, 48.79, 48.70, 46.49, 41.92, 39.44, 37.81, 35.45, 35.22, 35.10,29.73, 29.63, 28.89, 28.33, 27.50, 27.34, 23.39, 22.97, 22.92, 21.28,12.72; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M−H]⁺) 614.3798(24.5%), calcd. 614.3778.

Compound 106: Compound 124 (0.145 g, 0.236 mmol) was dissolved in CH₂Cl₂(2 mL) and MeOH (1 mL). 20% NaOH solution (0.2 mL) was added. Themixture was stirred for 12 hr, and anhydrous Na₂SO₄ was used to removewater. After concentration in vacuo, the residue was purified by silicagel chromatography (EtOAc/hexanes 1:3) to afford the desired product(0.124 g,92% yield) as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 4.29(bs, 1 H), 3.69-3.60 (m, 2 H), 3.52 (t, J=6.0 Hz, 2 H), 3.45-3.32 (m, 8H), 3.26 (d, J=2.7 Hz, 1 H), 3.17-3.02 (m, 2 H), 2.19-1.94 (m, 4 H),1.90-1.62 (series of multiplets, 13 H), 1.57-1.20 (series of multiplets,7 H), 0.97 (dd, J=14.3 and 3.1 Hz, 1 H), 0.90 (s, 3 H), 0.73 (s, 3 H);¹³C NMR (CDCl₃, 75 MHz) δ 79.69, 78.03, 75.47, 73.38, 65.46, 65.00,64.47, 48.87, 48.68, 46.83, 41.93, 39.71, 37.87, 35.43, 35.20, 35.09,29.96, 29.69, 29.59, 29.53, 28.89, 28.44, 27.48, 23.72, 22.91, 22.71,11.77. The alcohol (0.124 g, 0.216 mmol) was dissolved in dry THF (20mL) followed by the addition of LiAlH₄ (33 mg, 0.866 mmol). The graysuspension was stirred under N₂ for 12 hr. Na₂SO₄.10 H₂O (about 2 g) wascarefully added. After the gray color in the suspension dissipated,anhydrous Na₂SO₄ was added and the precipitate was removed byfiltration. After the removal of solvent, the residue was purified bycolumn chromatography (SiO₂, MeOH/CH₂Cl₂/28% NH₃.H₂O 2.5: 2.5:1). Afterconcentration of the relevant fractions, 1 M HC1(2 mL) was added todissolve the milky residue. The resulting clear solution was washed withEt₂O (2×10 mL). To the aqueous phase, 20% NaOH solution was added untilthe solution became strongly basic. CH₂Cl₂ (20 mL, 2×10 mL) was used toextract the basic solution. The combined extracts were dried overanhydrous Na₂SO₄ and removal of solvent gave the desired product (0.050g, 47% yield) as a colorless oil. ¹H NMR (20% CDCl₃ in CD₃OD, 300 MHz) δ4.77 (s, 7 H), 4.25 (t, J=8.5 Hz, 1 H), 3.75-3.68 (m, 1 H), 3.66-3.58(m, 1 H), 3.55 (t, J=6.1 Hz, 2 H), 3.48-3.41 (m, 1 H), 3.34 (bs, 1 H),3.30 (d, J=3.6 Hz, 1 H), 3.17-3.08 (m, 2 H), 2.86-2.70 (m, 6 H),2.20-1.91 (m, 4 H), 1.88-1.16 (series of multiplets, 19 H), 1.00 (dd,J=14.2 and 3.0 Hz, 1 H), 0.93 (s, 3 H), 0.73 (s, 3 H); ¹³C NMR (20%CDCl₃ in CD₃OD, 75 MHz) δ 80.62, 79.12, 76.74, 73.77, 68.50, 67.79,67.17, 47.69, 43.04, 40.76, 40.64, 40.62, 40.22, 39.01, 36.32, 36.25,35.94, 34.27, 33.97, 33.72, 30.13, 29.53, 28.43, 24.48, 23.58, 23.40,12.38; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e ([M+H]⁺) 496.4108 (100%),calcd. 496.4114.

Example 9

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 109 and 126-129.

Compound 126: Compound 125 (2.30 g, 3.52 mmol) was dissolved in MeOH (50mL) and CH₂Cl₂ (100 mL). A small amount of Et₃N was added, and thesolution was cooled to −78° C. Ozone was bubbled through the solutionuntil a blue color persisted. Me₂S (4 mL) was introduced followed by theaddition of NaBH₄ (0.266 g, 0.703 mmol) in MeOH (10 mL). The resultingsolution was allowed to warn and stir overnight. The solution wasconcentrated in vacuo, and brine (60 mL) was added. The mixture wasextracted with EtOAc (40 ml, 2×30 mL), and the combined extracts werewashed with brine and dried over anhydrous Na₂SO₄. Silica gelchromatography (EtOAc) afforded the product (1.24 g, 76% yield) as awhite solid. m.p. 219-220 C.; ¹H NMR (CDCl₃, 300 MHz) δ 5.10 (t, J=2.8Hz, 1 H), 4.90 (d, J=2.7 Hz, 1 H), 3.73-3.59 (m, 2 H), 3.56-3.44 (m, 1H), 2.13 (s, 3 H), 2.09 (s, 3 H), 2.07-0.95 (series of multiplets, 23H), 0.91 (s, 3 H), 0.83 (d, J=6.3 Hz, 3 H), 0.74 (s, 3 H); ¹³C NMR(CDCl₃, 75 MHz) δ 170.84, 170.82, 75.63, 71.77, 71.03, 60.73, 48.10,45.26, 43.54, 41.16, 38.78, 37.89, 35.00, 34.43, 32.26, 31.50, 30.60,29.07, 27.50, 25.70, 22.96, 22.71, 21.81, 21.63, 18.18, 12.35; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 465.3197 (20%), calcd. 465.3216.

Compound 127: Compound 126 (1.24 g, 2.67 mmol) was dissolved in MeOH (30mL), and NaOH (0.54 g, 13.4 mmol) was added. The suspension was refluxedunder N₂ for 24 hr. The MeOH was removed in vacuo followed by theaddition of H₂O (50 mL). The precipitate was filtered, washed with H₂Oand then dried in vacuo to give a white solid (1.02 g). This solid wasdissolved in DMF (40 mL) followed by the sequential addition of NEt₃(1.12 mL, 8.02 mmol), DMAP (16.3 mg, 0.13 mmol) and trityl chloride(1.49 g, 5.34 mmol). The suspension was stirred under N₂ for 12 hr andthen heated up to 50° C. for 24 hr. H₂O (100 mL) was added to the cooledsuspension, and the mixture was extracted with EtOAc (3×50 mL). Thecombined extracts were washed with brine (100 mL), dried over anhydrousNa₂SO₄, and concentrated in vacuo. Silica gel chromatography (EtOAc)afforded the product (1.20 g, 72% yield) as a pale yellow glass. To thisglass was added dry THF (80 mL) and NaH (60% in mineral oil, 0.77 g,19.3 mmol). The suspension was refluxed under N₂ for half an hour beforethe introduction of allylbromide (1.67 mL, 19.3 mmol). After 48 hr atreflux, another 10 eq. of NaH and allylbromide were introduced. Afteranother 48 hr, the reaction mixture was cooled and H₂O (100 mL) wasslowly added. The resulting mixture was extracted with hexanes (3×50mL), and the combined extracts were washed with brine (100 mL) and driedover anhydrous Na₂SO₄. Silica gel chromatography (5% EtOAc in hexanes)afforded the product (1.27 g, 64% yield for all three steps) as a clearglass. ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.43 (m, 6 H), 7.29-7.16 (m, 9 H),5.98-5.81 (m, 3 H), 5.29-5.18 (m, 3 H), 5.14-5.03 (m, 3 H), 4.11-3.97(m, 4 H), 3.75-3.67 (m, 2 H), 3.49 (bs, 1 H), 3.32-3.13 (d, J=2.4 Hz, 1H), 3.20-3.13 (m, 2 H), 3.00 (m, 1 H), 2.33-2.12 (m, 3 H), 2.03-0.92(series of multiplets, 19 H), 0.88 (s, 3 H), 0.78 (d, J=6.6 Hz, 3 H),0.65 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.71, 136.08, 136.04, 135.94,128.80, 127.76, 126.86, 116.30, 115.57, 86.53, 80.77, 79.20, 74.96,69.42, 69.34, 68.81, 62.00, 46.87, 46.48, 42.67, 42.11, 39.90, 36.15,35.50, 35.14, 35.10, 33.23, 28.99, 28.09, 27.75, 27.56, 23.36, 23.32,23.12, 18.24, 12.66; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)765.4875 (100%), calcd. 765.4859.

Compound 128: To a THF (40 mL) solution of 127 (1.27 g, 1.71 mmol) wasadded 9-BBN (0.5 M solution in THF, 17.1 mL). The mixture was stirredfor 12 hr before the addition of NaOH (20% solution, 10 mL) and H₂O (30%solution, 10 mL). The resulted mixture was refluxed for 1 hr followed bythe addition of brine (100 mL) and extraction with EtOAc (4×30 mL). Thecombined extracts were dried over anhydrous Na₂SO₄ and concentrated invacuo. Silica gel chromatography (5% MeOH in CH₂Cl₂) afforded theproduct (1.26 g, 93% yield) as a clear glass. ¹H NMR (5% CD₃OD in CDCl₃,300 MHz) δ 7.46-7.43 (m, 6 H), 7.32-7.20 (m, 9 H), 3.94 (s, 3 H),3.78-3.56 (m, 10 H), 3.48 (bs, 1 H), 3.32-3.26 (m, 2 H), 3.24-3.12 (m, 3H), 3.00 (dd, J=8.2 and 6.1 Hz, 1 H), 2.23-1.96 (m, 3 H), 1.90-0.95(series of multiplets, 25 H), 0.90 (s, 3 H), 0.77 (d, J =6.6 Hz, 3 H),0.66 (s, 3 H); ¹³C NMR (5% CD₃OD in CDCl₃, 75 MHz) δ 144.52, 128.64,127.64, 126.76, 86.43, 80.55, 79.31, 77.65, 77.23, 76.80, 76.06, 66.17,66.01, 65.41, 61.93, 61.20, 60.73, 60.39, 47.29, 46.08, 42.65, 41.62,39.49, 36.02, 35.10, 34.89, 34.77, 32.89, 32.71, 32.41, 32.26, 28.68,27.70, 27.51, 27.19, 23.26, 22.66, 22.50, 18.23, 12.34; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 819.5169 (100%), calcd.819.5099.

Compound 129: To a CH₂Cl₂ (50 mL) solution of compound 128 (1.26 g, 1.58mmol) at 0° C. was added Et₃N (0.92 mL, 6.60 mmol) followed by mesylchloride (0.47 mL, 6.05 mmol). After 15 minutes, H₂O (10 mL) wasfollowed by brine (80 mL). The mixture was extracted with EtOAc (60 mL,2×30 mL) and the combined extracts were dried over anhydrous Na₂SO₄.After removal of solvent in vacuo, the residue was dissolved in DMSO (10mL) and NaN₃ (1.192 g, 18.3 mmol) was added. The suspension was heatedto 60° C. under N₂ overnight. H₂O (100 mL) was added, and the mixturewas extracted with EtOAc (3×40 mL). The combined extracts were washedwith brine and dried over anhydrous Na₂SO₄. Removal of the solvent invacuo afforded a pale yellow oil. The oil was dissolved in MeOH (10 mL)and CH₂Cl₂ (20 mL) and TsOH (17.4 mg, 0.092 mmol) was added. After 12hr, saturated aqueous NaHCO₃ (20 mL) and brine (50 mL) were added andthe mixture was extracted with EtOAc (3×40 mL). The combined extractswere washed with brine (50 mL) and dried over anhydrous Na₂SO₄. Silicagel chromatography (EtOAc/hexanes 1:3) afforded the desired product(0.934, 94%) as a pale yellow oil. ¹H NMR (CDCl₃, 500 MHz) δ 3.75-3.70(m, 1 H), 3.68-3.63 (m, 2 H), 3.62-3.57 (m, 1 H), 3.53 (t, J=6.1 Hz, 2H), 3.50 (bs, 1 H), 3.46-3.38 (m, 6 H), 3.26 (d, J=2.4 Hz, 1 H),3.24-3.20 (m, 1 H), 3.16-3.12 (m, 1 H), 3.10-3.04 (m, 1 H), 2.17-2.04(m, 3 H), 1.96-1.63 (m, 14 H), 1.53-1.45 (m, 3 H), 1.35-1.20 (m, 7 H),1.08-1.00 (m, 1 H), 0.97-0.88 (m, 1 H), 0.94 (d, J=6.8 Hz, 3 H), 0.89(s, 3 H), 0.67 (s, 3 H); ¹³ C NMR (CDCl₃, 75 MHz) δ 80.64, 79.81, 76.06,65.05, 64.49, 64.34, 61.03, 49.02, 48.98, 48.78, 46.93, 46.53, 42.76,42.01, 39.83, 39.14, 35.46, 35.33, 35.12, 32.97, 29.79, 29.73, 29.10,27.90, 27.68, 23.56, 23.06, 22.88, 18.24, 12.60; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 652.4285 (100%), calcd.652.4295.

Compound 109: Compound 129 (0.245 g, 0.391 mmol) was dissolved in THF(30 mL) followed by the addition of LiAlH₄ (59 mg, 1.56 mmol). The graysuspension was stirred under N₂ 12 hr. Na₂SO₄.10H₂O powder (about 1 g)was carefully added. After the gray color in the suspension dissipated,anhydrous Na₂SO₄ was added and the precipitate was removed byfiltration. After the removal of solvent, the residue was purified bysilica gel chromatography (CH₂Cl₂/MeOH/28% NH₃.H₂O 10: 5:1 then10:5:1.5). The solvent was removed from relevant fractions, and 1 M HCl(4 mL) was added to dissolve the residue. The resulting clear solutionwas extracted with Et₂O (3×10 mL). 20% NaOH solution was added until thesolution became strongly basic. CH₂Cl₂ (4×10 mL) was used to extract thebasic solution. The combined extracts were dried over anhydrous Na₂SO₄,and removal of solvent in vacuo gave the desired product (0.15 g, 71%yield) as a colorless oil. ¹H NMR (about 20% CD₃OD in CDCl₃, 500 MHz) δ4.73 (bs, 7 H), 3.74-3.70 (m, 1 H), 3.65-3.60 (m, 2 H), 3.56-3.52 (m, 4H), 3.31-3.28 (m, 2 H), 3.16-3.09 (m, 2 H), 2.82-2.71 (m, 6 H),2.19-2.06 (m, 3 H), 1.97-1.66 (series of multiplets, 15 H), 1.58-1.48(m, 3 H), 1.38-0.98 (m, 7 H), 0.96 (d, J=6.8 Hz, 3 H), 0.93 (s, 3 H),0.71 (s, 3 H), ¹³C NMR (about 20% CD₃OD in CDCl₃, 75 MHz) δ 81.80,80.60, 77.17, 67.88, 67.86, 67.18, 60.73, 48.11, 47.28, 43.93, 42.99,41.34, 40.76, 40.72, 40.24, 39.70, 36.33, 36.18, 35.86, 34.29, 33.99,33.96, 33.83, 29.60, 29.00, 28.57, 28.54, 24.33, 23.59, 23.48, 18.86,13.04; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 552.4756 (100%),calcd. 552.4772.

Example 10

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 108 and 130.

Compound 130: o-NO₂C₆H₄SeCN (0.094 g, 0.21 mmol) and Bu₃P (0.095 mL,0.38 mmol) were stirred in dry THF (5 mL) at 0° C. for ½ hr followed bythe addition of compound 129 (0.10 g, 0.159 mmol) in THF (2 mL). Thesuspension was stirred for 1 hr followed by the addition of H₂O₂ (30%aqueous solution, 2 mL). The mixture was stirred for 12 hr followed byextraction with hexanes (4×10 mL). The combined extracts were dried overanhydrous Na₂SO₄. The desired product (0.035 g, 36% yield) was obtainedas pale yellowish oil after silical gel chromatography (10%EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 5.73-5.66 (ddd, J=17.1, 10.2,8.3 Hz, 1 H), 4.90 (dd, J=17.1, 2.0 Hz, 1 H), 4.82 (dd, J=10.2 Hz, 1.96Hz, 1 H), 3.68-3.64 (m, 1 H), 3.62-3.58 (m, 1 H), 3.54-3.26 (m, 9 H),3.25-3.22 (m, 2 H), 3.15-3.11 (m, 1 H), 3.10-3.04 (m, 1 H), 2.17-1.62(series of multiplets, 18 H), 1.51-1.43 (m, 2 H), 1.35-1.18 (m, 4 H),1.06-0.91 (m, 2 H), 1.02 (d, J−6.3 Hz, 3 H), 0.90 (s, 3 H), 0.68 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 145.50, 111.72, 80.60, 79.82, 76.09,65.06, 64.50, 64.45, 49.05, 48.97, 48.79, 46.43, 46.13, 42.76, 42.03,41.30, 39.84, 35.49, 35.34, 35.15, 29.82, 29.80, 29.75, 29.11, 28.00,27.84, 27.68, 23.56, 23.08, 22.95, 19.79, 12.87; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 634.4167 (90.6%), calcd.634.4169.

Compound 108: Compound 130 (0.105 g, 0.172 mmol) was dissolved in CH₂Cl₂(5 mL) and MeOH (5 mL) at −78° C. O₃ was bubbled into the solution forca. 20 min. Me₂S (1 mL) was added followed, and the solvent was removedin vacuo. The residue was dissolved in THF (15 mL), and LiAlH₄ (0.033 g,0.86 mmol) was added. The suspension was stirred for 12 hr. Na₂SO₄.10H₂O(about 2 g) was carefully added. After the gray color of the suspensiondissipated, anhydrous Na₂SO₄ was added and the precipitate was removedby filtration. Concentration and silica gel chromatography(CH₂Cl₂/MeOH/28% NH₃.H₂O10: 5:1.5 then 9:6:1.8) yielded a white glass.To this material was added 1 M HCl (4 mL). The resulting clear solutionwas washed with Et₂O (3×10 mL). 20% NaOH solution was added to theaqueous phase until the solution became strongly basic. CH₂Cl₂ (4×10 mL)was used to extract the basic solution. The combined extracts were driedover anhydrous Na₂SO₄ and removal of solvent gave the desired product(0.063 g, 68% yield) as a colorless oil. ¹H NMR (about 10% CD₃OD inCDCl₃, 500 MHz) δ 4.76 (bs, 7 H), 3.75-3.71 (m, 1 H), 3.66-3.62 (m, 1H), 3.58-3.52 (m, 4 H), 3.33-3.29 (m, 2 H), 3.22 (dd, J=10.5 and 7.6 Hz,1 H), 3.15-3.09 (m, 2 H), 2.81 (t, J=6.8 Hz, 2 H), 2.76-2.71 (m, 4 H),2.19-2.08 (m, 3 H), 2.00-1.66 (series of multiplets, 14 H), 1.58-1.45(m, 3 H), 1.40-1.08 (m, 5 H), 1.03 (d, J=6.8 Hz, 3 H), 1.02-0.96 (m, 1H), 0.93 (s, 3 H), 0.72 (s, 3 H); ¹³C NMR (about 10% CD₃OD in CDCl₃, 75MHz) δ 81.74, 80.64, 77.23, 67.95, 67.87, 67.18, 47.32, 44.59, 43.72,43.01, 41.26, 40.80, 40.71, 40.23, 40.02, 36.36, 36.20, 35.87, 34.27,33.99, 33.90, 29.60, 29.05, 28.58, 28.08, 24.49, 23.62, 23.46, 16.84,13.12; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 538.4578 (4.7%),calcd. 538.4584.

Example 11

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds CSA-21, 133-134 and CSA-15.

Compound CSA-21: Compound 115 (0.118 g, 0.183 mmol) was dissolved in dryCH₂Cl₂ (10 mL), and SO₃ pyridine complex (0.035 g, 0.22 mmol) was added.The suspension was stirred for 12 hr. The solvent was removed in vacuoto give white powder. To the white powder was added 1 M HCl (10 mL) andthe resulting mixture was extracted with CH₂Cl₂ (4×10 mL). The combinedextracts were dried over anhydrous Na₂SO₄. The desired product (0.11 g,84%) was obtained as a pale yellow oil after silica gel chromatography(10% MeOH in CH₂Cl₂). ¹H NMR (about 10% CD₃OD in CDCl₃, 500 MHz) δ 4.03(t, J=6.8 Hz, 2 H), 3.69-3.65 (m, 1 H), 3.62-3.58 (m, 1 H), 3.55 (t,J=6.1 Hz, 2 H), 3.51 (bs, 1 H), 3.46-3.38 (m, 6 H), 3.27 (d, J=2.4 Hz, 1H), 3.26-3.21 (m, 1 H), 3.18-3.07 (m, 2 H), 2.18-2.03 (m, 3 H),1.95-1.47 (series of multiplets, 19 H), 1.40-0.96 (series of multiplets,9 H), 0.92 (d, J=6.8 Hz, 3 H), 0.91 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR(about 10% CD₃OD in CDCl₃, 75 MHz) δ 80.43, 79.68, 75.87, 69.30, 64.82,64.32, 64.14, 48.78, 48.73, 48.50, 46.44, 46.21, 42.49, 41.76, 39.61,35.36, 35.17, 35.06, 34.85, 31.73, 29.53, 29.46, 29.44, 28.84, 27.68,27.48, 27.38, 25.91, 23.30, 22.75, 22.66, 17.70, 12.32; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M-H+2Na]⁺) 768.3831 (100%), calcd.768.3843. The azides were reduced by treating the triazide (0.11 g, 0.15mmol) with Ph₃ P (0.20 g, 0.77 mmol) in THF (10 mL) and H₂O (1 mL). Themixture was stirred for 3 days. The solvent was removed in vacuo, andthe residue was purified by silica gel chromatography (CH₂Cl₂/MeOH/28%NH₃.H₂O 12:6:1 then 10: 5:1.5) to afford the desired product (0.077 g,78% yield) as a glass. HCl in Et₂O (1 M, 0.5 mL) was added to the glassto give the corresponding HCl salt. ¹H NMR (about 10% CDC13 in CD₃OD,500 MHz) δ 4.81 (s, 10 H), 4.07-3.97 (m, 2 H), 3.82 (bs, 1 H), 3.71 (bs,1 H), 3.65 (t, J=5.2 Hz, 2 H), 3.57 (bs, 1 H), 3.37-3.30 (m, 2 H),3.22-3.02 (m, 8 H), 2.12-1.71 (series of multiplets, 17 H), 1.65-1.01(series of multiplets, 13 H), 0.97 (d, J=6.8 Hz, 3 H), 0.94 (s, 3 H),0.73 (s, 3 H); ¹³C NMR (about 10% CDCl₃ in CD₃OD, 75 MHz) δ 81.89,80.58, 77.50, 70.04, 66.71, 66.56, 66.02, 47.11, 46.76, 44.20, 42.66,40.50, 39.60, 39.40, 36.24, 36.11, 35.89, 35.67, 32.28, 29.38, 29.23,29.10, 28.94, 28.49, 26.06, 24.21, 23.46, 23.30, 18.50, 12.86; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 668.4271 (100%), calcd.668.4258.

Compound CSA-13: The mesylate derived from 23 (0.19 g, 0.264 mmol) wasstirred with excess octyl amine (2 mL) at 80° C. for 12 hr. Afterremoval of octylamine in vacuo, the residue was chromatographed (silicagel, EtOAc/hexanes 1:4 with 2% Et₃ N) to afford the desired product(0.19 g, 95% yield) as a pale yellow oil. ¹H NMR (CDCl₃, 300 MHz) δ3.69-3.37 (series of multiplets, 11 H), 3.26-3.00 (m, 4 H), 2.61-2.53(m, 4H), 2.20-2.02 (m, 3 H), 1.98-0.99 (series of multiplets, 40 H),0.92-0.85 (m, 9 H), 0.65 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.60,79.74, 76.05, 64.97, 64.40, 64.28, 50.79, 50.25, 49.00, 48.90, 48.71,46.47, 46.34, 42.65, 41.96, 39.80, 35.77, 35.41, 35.27, 35.05, 33.73,31.96, 30.25, 29.76, 29.74, 29.67, 29.39, 29.05, 27.84, 27.61, 27.55,26.70, 23.50, 23.00, 22.82, 22.79, 18.06, 14.23, 12.54; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 755.6012 (100%), calcd.755.6024. The triazide (0.18 g, 0.239 mmol) was dissolved in THF (10 mL)and EtOH (10 mL). Lindlar catalyst (44 mg) was added, and the suspensionwas shaken under H₂ (50 psi) for 12 hr. After removal of the solvent invacuo, the residue was purified by silica gel chromatography(CH₂Cl₂/MeOH/28% NH₃.H₂O 10:5:1, then 10:5:1.5). To the product, 1 M HCl(2 mL) and the resulting clear solution was extracted with Et₂O (2×10mL). 20% NaOH solution was added until the solution became stronglybasic. CH₂Cl₂ (20 mL, 2×10 mL) was used to extract the basic solution.The combined extracts were dried over anhydrous Na₂SO₄, and removal ofsolvent in vacuo gave the desired product (0.114 g, 68% yield) as aclear oil. ¹H NMR (about 20% CDCl₃ in CD₃OD, 500 MHz) δ 4.79 (bs, 7 H),3.74-3.70 (m, 1 H), 3.61 (m, 1 H), 3.56-3.51 (m, 3 H), 3.31-3.29 (m, 2H), 3.16-3.09 (m, 2 H), 2.88-2.72 (m, 6 H), 2.59-2.51 (m, 4 H),2.18-2.07 (m, 3 H), 1.97-1.66 (series of multiplets, 14 H), 1.62-0.97(series of multiplets, 25 H), 0.95 (d, J=6.3 Hz, 3 H), 0.93 (s, 3 H),0.89 (t, J=6.8 Hz, 3 H), 0.70 (s, 3 H); ¹³C NMR (about 20% CDCl₃ inCD₃OD, 75 MHz) δ 81.82, 80.63, 77.23, 67.85, 67.19, 51.20, 50.69, 47.82,47.24, 43.92, 43.01, 41.30, 40.80, 40.68, 40.22, 36.74, 36.38, 36.20,35.87, 34.66, 34.15, 33.87, 32.90, 30.54, 30.39, 30.30, 29.64, 29.03,28.59, 28.41, 26.96, 24.37, 23.65, 23.48, 18.75, 14.63, 13.09; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 677.6309 (46.6%), calcd.677.6309.

Compound CSA-46: Compound CSA-46 was prepared using the methods ofCSA-13, substituting 7-deoxycholic steroid backbone precursor in placeof cholic acid.

Compound 134: Compound CSA-13 (0.08 g, 0.12 mmol) was dissolved in CHCl₃(5 mL) and MeOH (5 mL), aminoiminosulfonic acid (0.045 g, 0.36 mmol) wasadded, and the suspension was stirred for 12 hr. The solvent was removedin vacuo, and the residue was dissolved in 1 M HCl (6 mL) and H₂O (10mL). The solution was washed with Et₂O (3×5 mL), and 20% NaOH solutionwas then added dropwise until the solution became strongly basic. Thebasic mixture was extracted with CH₂CT₂ (4×5 mL). The combined extractswere dried over anhydrous Na₂SO₄ and concentrated in vacuo to give thedesired product (0.087 g, 91% yield) as a white glass. ¹H NMR (about 20%CDCl₃ in CD₃OD, 500 MHz) δ 4.96 (bs, 13 H), 3.74-3.68 (m, 1 H),3.65-3.50 (m, 4 H), 3.38-3.18 (series of multiplets, 10 H), 2.60-2.50(m, 4 H), 2.15-1.99 (m, 3 H), 1.88-1.72 (m, 14 H), 1.60-0.99 (series ofmultiplets, 25 H), 0.94 (bs, 6 H), 0.89 (t, J=6.6 Hz, 3 H), 0.71 (s, 3H); ¹³C NMR (about 20% CDCl₃ in CD₃OD, 75 MHz) δ 159.00, 158.87, 158.72,81.68, 79.93, 76.95, 66.59, 65.93, 65.45, 50.82, 50.40, 47.64, 46.94,43.67, 42.27, 40.18, 39.25, 36.19, 35.66, 35.40, 34.21, 32.45, 30.51,30.26, 30.18, 30.10, 29.86, 29.35, 28.71, 28.15, 28.00, 26.87, 23.94,23.44, 23.23, 23.12, 18.61, 14.42, 12.98; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+H]⁺) 803.6958 (18.4%), calcd. 803.6953.

Compound CSA-15: The mesylate derived from 23 (0.092 g, 0.128 mmol) wasdissolved in DMSO (2 mL) followed by the addition of NaN₃ (0.0167 g,0.256 mmol). The suspension was heated to 70° C. for 12 hr. H₂O (20 mL)was added to the cooled suspension, and the mixture was extracted withEtOAc/hexanes (1:1) (20 mL, 3×10 mL). The combined extracts were washedwith brine (30 mL), dried over anhydrous Na₂SO₄, and concentrated invacuo to give the product (0.081 g, 95% yield) as a pale yellow oil. ¹HNMR (CDCl₃, 300 MHz) δ 3.69-3.36 (m, 11 H), 3.25-3.02 (m, 6 H),2.20-2.02 (m, 3 H), 1.97-1.60 (m, 15 H), 1.55-0.98 (m, 13 H), 0.92 (d,J=6.3 Hz. 3 H), 0.89 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ80.59, 79.77, 76.03, 65.01, 64.46, 64.30, 52.12, 48.99, 48.95, 48.76,46.44, 46.42, 42.70, 41.99, 39.82, 35.56, 35.44, 35.31, 35.09, 33.09,29.79, 29.77, 29.71, 29.08, 27.88, 27.78, 27.66, 25.65, 23.53, 23.03,22.85, 18.00, 12.58; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)691.4512 (100%), calcd. 691.4496. The tetraazide (0.081 g, 0.12 mmol)was dissolved in THF (5 mL) and EtOH (10 mL). Lindlar catalyst (30 mg)was added, and the suspension was shaken under H₂ (50 psi) for 12 hr.After removal of the solvent in vacuo, the residue was purified bysilica gel chromatography (CH₂Cl₂/MeOH/28% NH₃.H₂O 5:3:1, then 2:2:1).To the product, 1M HCl (2 mL) was added, and the resulting solution waswashed with Et₂O (2×10 mL). 20% NaOH solution was added to the aqueousphase until the solution became strongly basic. CH₂Cl₂ (10 mL, 2×5 mL)was used to extract the basic solution. The combined extracts were driedover anhydrous Na₂SO₄, and concentration in vacuo gave the desiredproduct (0.044 g, 64% yield) as a colorless oil. ¹H NMR (about 20% CDCl₃in CD₃OD, 500 MHz) δ 4.79 (bs, 8 H), 3.74-3.70 (m, 1 H), 3.66-3.62 (m, 1H), 3.56-3.52 (m, 3 H), 3.31-3.27 (m, 2 H), 3.16-3.10 (m, 2 H),2.82-2.70 (m, 6 H), 2.64-2.54 (m, 2 H), 2.19-2.07 (m, 3 H), 1.99-1.66(series of multiplets, 14 H), 1.58-0.96 (series of multiplets, 13 H),0.96 (d, J=6.6 Hz, 3 H), 0.93 (s, 3 H), 0.70 (s, 3 H); ¹³C NMR (about20% CDCl₃ in CD₃OD, 75 MHz) δ 81.96, 90.76, 77.33, 67.92, 67.26, 47.84,47.33, 44.04, 43.24, 43.15, 41.40, 40.91, 40.78, 40.29, 36.82, 36.48,36.28, 35.96, 34.39, 34.11, 30.59, 29.69, 29.13, 28.68, 28.64, 24.43,23.69, 23.48, 18.77, 13.06; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+H]⁺) 565.5041 (100%), calcd. 565.5057.

Example 12

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 203a-b, 207a-c, 209a-c, 210a-b andCSA-31.

Compounds 203a-b, 207a-c, 208a-c, 209a-c, and 210a-b: BOC-glycine wasreacted with DCC, DMAP and cholic acid derivative 201 (Scheme 11) togive triester 202a in good yield. A similar reaction incorporatingBOC-β-alanine was also successful, giving 202b. Deprotection of 202a and202b with HCl in dioxane, followed by purification (SiO₂ chromatographywith a CH₂Cl₂ MeOH/NH₄OH eluent), gave triesters 203a and 203b in goodyield.

Triamides of glycine and β-alanine (207a and 207b, respectively) wereformed using the same reaction conditions (Scheme 12). Triamides witha-branched amino acids could also be formed. For example, under theconditions described, a triamide with bis-BOC-lysine side chains wasformed (compound 207c). The C24 esters of 207a-c were hydrolyzed withLiOH in THF and methanol to give alcohols 208a-c. Deprotection using HClin dioxane (208a-c) gave triamides 209a-c in good yield. In addition,alcohols 208a and 208b were mesylated and reacted with benzylmethylamine. Deprotection of the resulting compounds with HCl in dioxane gavetriamides 210a and 210b (Scheme 12). Compound CSA-31 was prepared byanalogy to compounds 210a and 210b.

Example 13

This example includes a description of one or more exemplary syntheticprocedures for obtaining Compounds 302, 312-321, 324-326, 328-331 and341-343.

Compound 302: Compound 308 (5β-cholanic acid 3,7,12-trione methyl ester)was prepared from methyl cholate and pyridinium dichromate in nearquantitative yield from methyl cholate. Compound 308 can also beprepared as described in Pearson et al., J. Chem. Soc. Perkins Trans.11985, 267; Mitra et al., J. Org. Chem. 1968, 33, 175; and Takeda etal., J. Biochem. (Tokyo) 1959, 46, 1313. Compound 308 was treated withhydroxyl amine hydrochloride and sodium acetate in refluxing ethanol for12 hr (as described in Hsieh et al., Bioorg. Med. Chem. 1995, 3, 823),giving 309 in 97% yield.

A 250 ml three neck flask was charged with glyme (100 ml); to this wasadded 309 (1.00 g, 2.16 mmol) and sodium borohydride (2.11 g, 55.7mmol). TiCl₄ (4.0 mL, 36.4 mmol) was added to the mixture slowly undernitrogen at 0° C. The resulting green mixture was stirred at roomtemperature for 24 hours and then refluxed for another 12 h. The flaskwas cooled in an ice bath, and ammonium hydroxide (100 mL) was added.The resulting mixture was stirred for 6 hours at room temperature. Conc.HCl (60 mL) was added slowly, and the acidic mixture was stirred for 8hours. The resulting suspension was made alkaline by adding solid KOH.The suspension was filtered and the solids were washed with MeOH. Thecombined filtrate and washings were combined and concentrated in vacuo.The resulting solid was suspended in 6% aqueous KOH (100 mL) andextracted with CH₂Cl₂ (4×75 mL). The combined extracts were dried overNa₂SO₄ and solvent was removed in vacuo to give 1.14 g of a white solid.The mixture was chromatographed on silica gel (CH₂Cl₂/MeOH/NH₄OH 12:6:1)giving 302 (0.282 g, 33% yield), 3 (0.066 g, 8% yield), 4 (0.118 g, 14%yield).

Compound 302: m.p. 200-202° C.; ¹H NMR (about 10% CDCl₃ in CD₃OD, 300MHz) δ 4.81 (bs, 7 H), 3.57-3.49 (m, 2 H), 3.14 (t, J=3.2 Hz, 1 H), 2.97(bs, 1 H), 2.55-2.50 (m, 1 H), 2.15-2.10 (m, 1 H), 1.95-1.83 (m, 3 H),1.74-0.99 (series of multiplets, 20 H), 1.01 (d, J=6.4 Hz, 3 H), 0.95(s, 3 H), 0.79 (s, 3 H); ¹³C NMR (10% CDCl₃ in CD₃OD, 75 MHz) δ 63.28,55.01, 52.39, 49.20, 48.69, 47.00, 43.24, 42.77, 41.03, 40.27, 36.82,36.35, 35.75, 35.12, 32.77, 31.36, 30.10, 28.54, 27.88, 26.96, 24.35,23.38, 18.18, 14.23, HRFAB-MS (thioglycerol+Na⁺ matrix) m/e; ([M+H]⁺)392.3627 (100%); calcd. 392.3641.

Octanyl cholate (328): Cholic acid (3.14 g, 7.43 mmol) and10-camphorsulfonic acid (0.52 g, 2.23 mmol) were dissolved in octanol(3.5 mL, 23.44 mmol). The solution was warmed to 40-50° C. in oil bathunder vacuum (about 13 mm/Hg). After 14 h, the remaining octanol wasevaporated under high vacuum. The crude product was purified viachromatography (silica gel, 5% MeOH in CH₂Cl₂) to afford the desiredproduct (2.81 g, 73% yield) as a white powder. ¹H NMR (CDCl₃, 500 MHz) δ4.06 (t, J=6.7 Hz, 2 H), 3.98 (s, 1 H), 3.86 (s, 1 H), 3.48-3.44 (m, 1H), 2.41-2.34 (m, 1 H), 2.28-2.18 (m, 3 H), 1.98-1.28 (series ofmultiplets, 35 H), 0.99 (d, J=3.3 Hz, 3 H), 0.90 (s, 3 H), 0.89 (t, J=7Hz, 3 H), 0.69 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 154.38, 73.18, 72.14,68.63, 56.07, 50.02, 49.32, 47.07, 46.74, 41.96, 41.67, 39.84, 39.76,35.66, 35.45, 34.95, 34.86, 34.15, 32.97, 32.91, 31.65, 31.11, 30.68,28.39, 27.78, 26.66, 26.52, 25.82, 25.70, 25.54, 25.15, 24.95, 23.45,22.69, 17.77, 12.71; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)543.4015 (100%), calcd. 543.4026.

Representative synthesis of compounds 329-331: Octanyl cholate (328)(0.266 g, 0.511 mmol), N-t-Boc-glycine (0.403 g, 2.298 mmol), DCC (0.474g, 2.298 mmol) and DMAP (0.0624 g, 0.051 mmol) were mixed in CH₂Cl₂ (15mL) for 3 h. The resulting white precipitate was removed by filtration.The filtrate was concentrated, and the product was purified bychromatography (silica gel, EtOAc/Hexane 1:2) to afford the desiredproduct (0.481 g, 95% yield) as a white powder. Compound 329 ¹H NMR(CDCl₃, 300 MHz) δ 5.18 (br, 3 H), 5.01 (s, 1 H), 4.61 (m, 1 H), 4.04(t, J=6.5 Hz, 2 H), 3.97-3.88 (series of multiplets, 6 H), 2.39-2.15(series of multiplets, 2 H), 2.06-1.02 (series of multiplets, 35 H),1.46 (s, 18 H), 1.45 (s, 9 H), 0.93 (s, 3 H), 0.88 (t, J=6.7 Hz, 3 H),0.81 (d, J=6 Hz, 3 H), 0.74 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 174.26,170.19, 169.9, 169.78, 155.87, 155.67, 79.95, 76.47, 75.167, 72.11,64.55, 47.40, 45.28, 43.17, 42.86, 40.82, 37.94, 34.71, 34.63, 34.43,31.86, 31.340, 31.20, 30.76, 29.29, 29.25, 28.80, 28.72, 28.42, 28.06,27.96, 27.19, 26.81, 26.29, 26.012, 25.66, 22.87, 22.71, 22.57, 17.55,14.18, 12.27; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)1014.6261 (100%), calcd. 1014.6242. Compound 330: ¹H NMR (CDCl₃, 500MHz) δ 5.10 (s, 1 H), 4.92 (d, J=2.44 Hz, 1 H), 4.55 (m, 1 H), 4.00 (t,J=6.8 Hz, 2 H), 3.39-3.33 (series of multiplets, 6 H), 2.595-2.467(series of multiplets, 6 H), 2.31-2.12 (series of multiplets, 2 H),2.01-1.00 (series of multiplets, 37 H), 1.39 (s, 27 H), 0.88 (s, 3 H),0.84 (t, J=6.8 Hz, 3 H), 0.76 (d, J=6.3 Hz, 3 H), 0.69 (s, 3 H); ¹³C NMR(CDCl₃, 75 MHz) δ 174.16, 172.10, 171.78, 171.67, 155.95, 79.45, 75.67,74.21, 71.10, 64.63, 47.79, 45.27, 43.52, 40.97, 37.92, 36.35, 35.14,35.05, 34.90, 34.71, 34.46, 31.91, 31.45, 30.95, 29.35, 29.31, 28.96,28.78, 28.56, 28.55, 27.22, 26.98, 26.269, 25.71, 23.00, 22.77, 22.64,17.75, 14.24, 12.39; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)1056.6702 (100%), calcd. 1056.6712. Compound 331 ¹³C NMR (CDCl₃, 125MHz) δ 174.00, 172.75, 172.41, 172.30, 156.03, 79.00, 75.28, 73.79,70.77, 64.39, 47.43, 45.04, 43.21, 40.76, 40.00, 39.93, 37.78, 34.74,34.62, 34.23, 32.19, 32.01, 31.70, 31.24, 30.77, 29.13, 29.10, 28.67,38.58, 28.38, 25.86, 25.37, 22.56, 22.38, 17.51, 14.05, 12.13; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 1098.7181 (100%), calcd.1098.7181.

Representative synthesis of compounds 341-343: To compound 329 (0.463 g,0.467 mmol) was added HCl in dioxane (0.3 mL, 4.0 M). After stirring themixture for 30 min, the excess HCl and solvent were removed in vacuo.The product was isolated, after chromatography (silica gel,CH₂Cl₂/MeOH/NH₃.H₂O 10:1.2:0.1) as a (0.271 g, 84%) pale oil. Thetrihydrochloride salt of 341 was prepared by addition of HCl in dioxaneand evaporation of excess HCl and dioxane in vacuo giving a whitepowder. Compound 341: ¹H NMR (CDCl₃ with about 10% CD₃OD, 500 MHz) δ5.16 (s, 1 H), 4.99 (t, J=3.6 Hz, 1 H), 4.61 (m, 1 H), 4.04 (t, J=6.8Hz, 2 H), 3.51-3.36 (m, 6 H), 2.34-2.15 (m, 2 H), 2.00-1.05 (series ofmultiplets, 40 H), 0.93 (s, 3 H), 0.88 (t, J=7.1 Hz, 3 H), 0.80 (d,J=3.2 Hz, 3 H), 0.74 (s, 3 H); ¹³C NMR (CDCl₃ and about 10% CD₃OD, 75MHz) δ 174.32, 173.92, 173.81, 76.08, 74.67, 71.61, 64.73, 47.64, 45.39,44.41, 43.49, 40.97, 37.99, 34.99, 34.77, 34.71, 34.52, 31.96, 31.54,31.35, 30.96, 29.39, 29.36, 29.02, 28.82, 27.32, 27.11, 26.11, 25.83,23.01, 22.82, 22.69, 17.79, 14.28, 12.41; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 714.4651 (100%), calcd. 714.4669.

Compound 342: ¹H NMR (CDCl₃ and about 10% CD₃OD, 300 MHz) δ 5.142 (s, 1H), 4.96 (d, J=2.7 Hz, 1 H), 4.60, (m, 1 H), 4.04 (t, J=6.6 Hz, 2 H),3.07-2.95 (series of multiplets, 6 H), 2.56-2.43 (series of multiplets,6 H), 2.38-2.13 (series of multiplets, 2 H), 2.07-1.02 (series ofmultiplets, 36 H), 0.92 (s, 3 H), 0.88 (t, J=6.6 Hz, 3 H), 0.82 (d,J=6.6 Hz, 3 H), 0.73 (s, 3 H); ¹³C NMR (CDCl₃ and CD₃OD, 75 MHz) δ174.29, 172.2 171.98, 171.92, 75.52, 74.09, 70.98, 64.67, 47.78, 45.26,43.52, 40.98, 38.73, 38.62, 38.35, 38.07, 38.03, 37.99, 35.01, 34.81,34.77, 34.49, 31.92, 31.50, 31.40, 30.99, 29.36, 29.33, 28.93, 28.80,27.43, 26.96, 26.08, 25.56, 23.07, 22.79, 22.62, 17.73, 14.25, 12.34;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 714.4651 (100%),calcd. 714.4669. Compound 343: ¹H NMR (CDCl₃ and CD₃OD, 500 MHz) δ 5.12(s, 1 H) 4.93 (s, 1 H), 4.59 (m, 1 H), 4.04 (t, J=7 Hz, 2 H), 2.79-2.69(series of multiplets, 6 H), 2.4621-2.2999 (series of multiplets, 6 H),2.2033-1.0854 (series of multiplets, 42 H), 0.94 (s, 2 H), 0.91 (s, 1H), 0.88 (t, J=7 Hz, 3 H), 0.82 (d, J=6.4 Hz, 3 H), 0.75 (s, 3 H), ¹³CNMR (CDCl₃ and CD₃OD, 75 MHz) δ 174.70, 171.97, 171.86, 171.75,76.10,74.55, 71.56, 64.85, 47.96, 45.31, 43.37, 40.87, 38.09, 34.86,34.80, 34.73, 34.46, 32.84, 32.62, 32.27, 31.87, 31.75, 31.42, 31.08,29.31, 29.28, 29.26, 28.78, 28.73, 27.38, 26.91, 26.05, 25.37, 23.24,23.15, 22.95, 22.74, 22.71, 22.43, 17.78, 14.11, 12.28; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 798.5624 (100%), calcd.798.5609.

Benzyl cholate (312): Cholic acid (4.33 g, 10.62 mmol) and10-caphorsulfonic acid (0.493 g, 2.21 mmol) were dissolved in benzylalcohol (1.97 mL, 19.3 mmol). The suspension was heated to 50° C. in oilbath and stirred under vacuum (about 13 mm/Hg) for 16 h. Excess benzylalcohol was removed in vacuo, and the crude product was chromatographed(silica gel, 5% MeOH in CH₂Cl₂) to give the desire product as a whitepowder (4.23 g, 81% yield). ¹H NMR (CDCl₃, 500 MHz) δ 7.34-7.33 (m, 5H), 5.10 (d, J=1.5 Hz, 2 H), 3.92 (s, 1 H), 3.81 (s, 1 H), 3.42 (s, 1H), 3.40 (br, m, 3 H), 2.44-2.38 (m, 1 H), 2.31-2.25 (m, 1 H), 2.219 (t,J=12 Hz, 2 H), 0.96 (d, J=5.5 Hz, 3 H), 0.86 (s, 3 H), 0.63 (s, 3 H);¹³C NMR (CDCl₃, 125 MHz) δ 174.25, 136.30, 128.66, 128.63, 128.32,128.28, 128.24, 73.18, 71.98, 68.54, 66.18, 47.14, 46.56, 41.69, 39.65,35.51, 35.37, 34.91, 34.84, 31.49, 31.08, 30.50, 28.31, 27.62, 26.47,23.35, 22.65, 22.60, 17.42, 12.63, 12.57; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 521.3235 (100%), calcd. 521.3242.

Representative synthesis of compounds 313-315: Benzyl cholate (312)(0.248 g, 0.499 mmol), N-t-Boc-glycine (0.404 g, 2.30 mmol), DCC (0.338g, 1.49 mmol) and DMAP (0.051 g, 0.399 mmol) were added to CH₂Cl₂ (15mL), and the suspension was stirred for 16 h. The resulting whiteprecipitate was removed by filtration, and the filtrate wasconcentrated. The product was obtained after chromatography (silica gel,EtOAc/Hexane 0.6:1) as a white powder (0.329 g, 68%). Compound 313: ¹HNMR (CDCl₃, 300 MHz) δ 7.34-7.33 (m, 5 H), 5.16 (s, 1 H), 5.08 (dd,J=22.5 Hz, 12.3 Hz, 4 H), 5.00 (s, 1 H), 4.60 (m, 1 H), 4.04-3.81(series of multiplets, 6 H), 2.43-1.01 (series of multiplets, 25 H),1.46 (s, 9 H), 1.44 (s, 18 H), 0.92 (s, 3 H), 0.797 (d, J=5.7 Hz, 3 H),0.69 (s, 1 H); ¹³C NMR (CDCl₃, 75 MHz) δ 173.99, 170.25, 170.05, 169.85,155.73, 136.19, 128.69, 128.45, 128.35, 80.06, 77.65, 77.23, 76.80,76.53, 75.24, 72.19, 66.29, 47.46, 45.35, 43.24, 42.91, 40.89, 38.00,34.79, 34.66, 34.49, 31.43, 31.25, 30.77, 28.88, 28.40, 27.23, 26.89,25.74, 22.94, 22.65, 17.61, 12.32; FAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 992.5468 (100%), calcd. 992.5460.

Representative synthesis of compounds 316-318: Compound 313 (0.505 g,0.520 mmol) and Pd (5 wt. % on active carbon, 0.111 g, 0.0521 mmol) wereadded to MeOH (5 mL). The suspension was stirred under H₂ (50 psi) for20 hours. The solids were removed by filtration and the filtrate wasconcentrated. Purification of the product via chromatography (silicagel, 5% MeOH in CH₂Cl₂) gave a white powder (0.450 g, 98% yield).Compound 316: ¹H NMR (CDCl₃, 500 MHz) δ 5.20 (s, 1 H), 5.12 (br., 2 H),4.92 (s, 1 H), 4.55 (m, 1 H), 3.98-3.83 (series of multiplets, 6 H),2.30-2.13 (series of multiplets, 2 H), 1.96-0.98 (series of multiplets,30 H), 1.40 (s, 9 H), 1.39 (s, 18 H), 0.87 (s, 3 H), 0.76 (d, J=6.3 Hz,3 H), 0.68 (s, 3 H); ¹³C NMR (CDCl₃ 75 MHz) δ 174.11, 165.60, 165.41,165.22, 151.28, 151.14, 75.48, 75.26, 71.81, 70.57, 67.50, 45.95, 42.58,40.65, 38.52, 38.16, 36.17, 33.28, 30.01, 29.78, 26.71, 26.42, 25.95,24.16, 23.78, 23.40, 23.31, 22.55, 22.16, 21.03, 18.23, 17.93, 12.91,7.61; FAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 902.4997 (21%),calcd. 902.4990.

Representative synthesis of compounds 319-321: Compound 316 (0.375 g,0.427 mmol), DCC (0.105 g, 0.512 mmol) and DMAP (0.062 g, 0.512 mmol)and N,N-dimethylethanolamine (0.09 ml, 0.896 mmol) were added to CH₂Cl₂(15 mL). The mixture for 16 h, and solvent and excessN,N-dimethylethanolamine were removed in vacuo. The product was purifiedvia chromatography (silica gel EtOAc/hexane/Et3 N, 12:10:0.6) giving awhite powder (0.330 g, 82% yield). ¹H NMR (CDCl₃ and about 10% CD₃OD,500 MHz) δ 5.18 (s, 1 H), 5.00 (s, 1 H), 4.19 (t, J=5.0 Hz, 2 H), 3.92(s, 3 H), 3.81 (s, 3 H), 2.62 (t, J=10 Hz, 2 H), 2.30 (s, 6 H), 1.47 (s,9 H), 1.47 (s, 1 H), 1.45 (s, 1 H), 2.12-1.05 (series of multiplets, 27H), 0.96 (s, 3 H), 0.84 (d, J=10.5 Hz, 3 H), 0.78 (s, 3 H); ¹³C NMR(CDCl₃ and about 10% CD₃OD, 125 MHz) δ 174.19, 170.05, 169.87, 156.21,79.36, 79.27, 76.06, 76.90, 71.80, 61.19, 57.04, 46.88, 44.87, 44.67,44.53, 42.78, 42.15, 42.01, 40.43, 37.47, 34.32, 34.11, 33.92, 33.35,33.25, 30.74, 30.56, 30.16, 28.40, 27.67, 27.62, 26.73, 26.19, 25.18,25.10, 24.72, 24.49, 22.29, 21.81, 16.76, 11.56; FAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 973.5723 (100%), calcd.973.5725. The white solid from the previous reaction (0.680 g, 0.714mmol) and MeI (1 M in CH₂Cl₂, 1.5 mL) were stirred together for 2 h. Thesolvent and excess MeI were removed in vacuo giving a white solid (0.812g about 100%). The product was carried on without further purification.

Representative synthesis of compounds 324-326: Compound 319 (0.812 g,0.714 mmol) was dissolved in CH₂Cl₂ (5 mL) and trifluoroacetic acid (0.5mL) was added. The mixture was stirred for 16 min. The solvent andexcess acid were removed in vacuo, and the resulting oil waschromatographed (silica gel, CH₂Cl₂/MeOH/NH₃.H₂O 4:4:1) to give thedesired product as a pale glass (0.437 g, 90% yield). Addition of HCl (2M in ethyl ether, 2.5 mL) gave the trihydrochloride salt of 324 as apale yellow powder.

Compound 324: ¹H NMR (50% CDCl₃, 50% CD₃OD, 300 MHz) δ 5.43 (s, 1 H),5.24 (s, 1 H), 4.84 (m, 1 H), 4.66 (m, 2 H), 4.16-3.96 (series ofmultiplets, 6 H), 3.88 (m, 2 H), 3.37 (s, 9 H), 0.67 (s, 3 H), 0.59 (d,J=6.3 Hz, 3 H), 0.56 (s, 3 H); ¹³C NMR (50% CDCl₃, 50% CD₃OD, 75 MHz) δ173.47, 167.06, 167.01, 166.70, 78.01, 76.49, 73.78, 64.98, 57.67,53.36, 47.49, 46.99, 45.61, 43.28, 40.83, 40.23, 40.10, 37.69, 34.80,34.48, 34.28, 31.03, 30.63, 30.44, 28.94, 27.05, 26.56, 25.50, 22.53,21.56, 16.95, 11.37; FAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M−I]⁺)665.4475 (85.6%), cacld 665.4489. Compounds 325 and 326 proved toounstable to chromatograph using the basic eluent used for thepurification of 324. Consequently, 325 and 326 were prepared bydeprotection of 320 and 321 using HC1(2 M in diethyl ether), followed bytituration with ethyl acetate. The compounds were then used withoutfurther purification. ¹H NMR spectroscopy indicated that compounds 325and 326 were >95% pure. Compound 325: ¹H NMR (50% CDCl₃, 50% CD₃OD, 500MHz) δ 5.21 (s, 1 H), 5.02 (d, J=4 Hz, 1 H), 4.64 (m, 1 H), 4.53 (m, 2H), 3.74 (m, 2 H), 3.31-3.01 (series of multiplets, 6 H), 3.23 (s, 9 H),2.96-2.73 (series of multiples, 6 H), 2.51-2.44 (m, 1 H), 2.35-2.29 (m,1 H), 2.14-1.09 (series of multiplets, 26 H), 0.99 (s, 3 H), 0.85 (d,J=6.5 Hz, 3 H), 0.80 (s, 3 H); ¹³C NMR (50% CDCl₃, 50% CD₃OD, 125 MHz) δ172.77, 169.88, 169.56, 169.50, 75.94, 74.44, 71.57, 64.31, 56.94,52.92, 46.78, 44.59, 42.70, 40.21, 37.16, 34.80, 34.72, 34.66, 34.05,34.00, 33.78, 33.62, 30.95, 30.91, 30.81, 30.41, 29.96, 29.81, 28.20,26.37, 26.06, 24.74, 24.24, 22.04, 21.13, 16.54, 10.97; FAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M−I]⁺) 707.4958 (25.6%), cacld707.4958. Compound 326: ¹H NMR (50% CDCl₃, 50% CD₃OD, 500 MHz) δ 5.12(s, 1H), 4.94 (d, J=2.5 Hz, 1 H), 4.56 (m. 1 H), 4.51 (t, J=2.3 Hz, 2H), 3.74 (m, 2 H), 3.23 (s, 9 H), 3.05-3.01 (m, 4 H), 2.98 (t, J=7.5 Hz,2 H), 2.63-2.43 (series of multiplets, 6 H), 2.31-2.24 (series ofmultiplets, 2 H), 2.07-1.87 (series of multiplets, 12 H), 1.17-1.05(series of multiplets, 23 H), 0.94 (s, 3 H), 0.82 (d, J=6.0 Hz, 3 H),0.76 (s, 3 H); ¹³C NMR (50% CDCl₃, 50% CD₃OD, 125 MHz) δ 171.87, 169.79,169.59, 169.50, 76.12, 74.70, 71.65, 65.57, 65.08, 64.40, 57.68, 53.74,52.78, 45.33, 43.54, 41.04, 39.12, 37.92, 43.85, 34.72, 34.56, 34.34,32.30, 31.47, 31.27, 30.87, 30.58, 29.03, 27.053, 26.84, 25.51, 24.95,24.91, 22.87, 22.82, 22.65, 21.93, 17.31, 11.81; FAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M−I]⁺) 749.5432 (100%), cacld 749.5436.

Example 14

This example includes data indicating the stability of Compounds 352-354under acidic, neutral and basic conditions.

Compounds 352-354 were dissolved in 50 mM phosphate buffered water (pH2.0, 7.0 or 12.0) at approximately 10 mM concentrations. The structuresof compounds 352-354 are given in FIG. 9. Decomposition of the compoundswas observed via HPLC (cyano-silica column, 0.15% TFA water-acetonitrilegradient elution). Table 15 shows the stabilities (half-lives) ofcompounds 352-354 in phosphate buffer at room temperature, pH 2.0, pH7.0 and pH 12.0. These compounds were used since they contain achromophore that facilitated monitoring of decomposition by absorptionmethods common in the HPLC apparatus used.

At low pH, the amines are expected to be protonated and the compoundsshowed relative stability. At higher pH, the amines were less stronglyprotonated and became involved in ester hydrolysis. The γ-aminobutyricacid-derived compound was especially susceptible to hydrolysis,presumably yielding pyrrolidone. In general, the compounds are believedto hydrolyse to give cholic acid, choline or octanol, and glycine,beta-alanine, or pyrrolidone, depending on the particular compound.

Decomposition through ester hydrolysis yielded compounds that were lesspolar and easily separable from the starting compounds. Initially, onlyone benezene-containing decomposition product was observed; at longerreaction times, two other decomposition products were observed whichpresumably corresponded to sequential ester hydrolysis,

Example 15

This example includes a description of additional exemplary syntheticprocedures for producing compounds of formula I. In one example,hydroxyl groups on cholic acid can be converted into amine groups asdescribed in in Hsich et al. (Synthesis and DNA Binding Properties ofC3-, C12-, and C24-Substituted Amino-Steroids Derived from Bile Acids,Biorganic and Medicinal Chemistry, 1995, vol. 6, 823-838).

Compounds of formula I prepared as shown in the following Scheme.

Example 16

This example describes various materials and methods.

Virus source and Culture: The Wyeth strain of vaccinia virus wasobtained from the Centers for Disease Control and Prevention. HeLa S3(ATCC#CCK-2.2) human adenocarcinoma cells were grown to confluence inRPMI media supplemented with 10% fetal calf sera (FCS) for use ofpropagation of vaccinia virus. The cells were rinsed and overlaid withRPMI-2.5% FCS then inoculated with 5×10⁶ PFU/T-175 flask and incubatedat 37° C. in 5% CO₂ for three days. Virus was harvested after disruptionof cells yielding infectious virions in the form of intracellular maturevirions (Schmelz, M. et al., Journal of Virology 68:130 (1994)).

Viral Plaque Assay: BS-C-1 (ATCC #CCL-26) African green monkey kidneycells were seeded at 2×10⁵/well in 24 well tissue culture plates inMEM-10% FCS, Pen/Strep and allowed to grow overnight before having thesupernatant removed and replaced with MEM-2.5% FCS for virus incubation.The BS-C-1 cells were used for the quantitative estimates because theypresent uniform plaques. HeLa S3 cells are routinely used forpreparations of virus stock as they give consistently high yields ofvirus, but due to their rounded morphology, do not present uniformplaques.

CSA compounds were diluted in 0.01× tryptic soy broth containing 10 mMsodium phosphate buffer, pH=7.4. Virus diluted in the same buffer wasadded to the compounds, and they were incubated for 24 hours at 37° C.Twenty microliters of the peptide/virus mixture was added to the cellsin 0.5 ml MEM-2.5% FCS and allowed to infect for 24 hours for RNAanalysis, or 48 hours for plaque development. For the plaque assay, themedium was removed and wells were overlaid with 0.5 ml 4% bufferedformalin, allowed to fix for 10 minutes at room temperature. Theformalin was removed and 0.5 ml 0.1% crystal violet in PBS was added tothe wells for 5 minutes at room temperature. Wells were then aspiratedand air-dried for visualization of plaques. Te most accurate resultswere obtained with the virus alone forming 50-80 plaques per well.

Keratinocyte Cell Culture: HaCaT cells, a human keratinocyte cell line,were cultured in Dulbecco's Modified Eagle's media (Cellgro)supplemented with 10% FCS (Gemini Bio Products) and 1% of the following:penicillin/streptomycin, L-glutamine, minimal essential medium withnon-essential amino acids (GIBCO), MEM vitamins solution (GIBCO) untilconfluent.

To investigate the anti-viral activity of CSA compounds, cells wereseeded in 24 well plates at a concentration of 2×10⁵ cells/well. Cellswere infected with vaccinia virus (0.05 plaque forming units/cell) for 6hours. Following the incubation, virus was removed and the cells werewashed with media to remove remaining virus. CSA compounds (0-100 μM)were added to the infected keratinocytes and allowed to incubate for anadditional 18 hours. RNA was isolated for analysis of vaccinia virusgene expression.

Vaccinia Gene Expression: RNA was isolated from cultured cells using theRNeasy Mini Kit (Qiagen, Valencia, Calif.) according to manufacturer'sguidelines. Real-time PCR was performed using an ABI 7000 SequenceDetection system (Applied Biosystems, Foster City, Calif.). The primersequences that were used to assay for the vaccinia gene transcripts are:Forward, 5′-GCCAATGAGGGTTCGAGTTC-3′ SEQ ID NO.:1 and Reverse,5′-CAACATCCCGTCGTTCATCA-3′ SEQ ID NO.:2. This region of the genomeencodes a subunit of a DNA-directed RNA polymerase expressed within twohours of viral entry (Amegadzie, B. Y. et al., Journal of BiologicalChemistry 266:13712 (1991)). The TaqMan probe was purchased from AppliedBiosystems: 5′ labeled with 6-carboxyfluorescein (FAM) and 3′-labeledwith 6-carboxy-tetramethylrhodamine (TAMRA). Amplification reactionswere performed in MicroAmp optical tubes (Applied Biosystems) in a 25 μlvolume containing 2× TaqMan Master Mix (Applied Biosystems), 900 nMforward primer, 900 nM reverse primer, 200 nM probe, and the templateRNA. Thermal cycling conditions were: 50° C. for 2 minutes, 95° C. for10 minutes for one cycle. Subsequently 40 cycles of amplification wereperformed at 94° C. for 15 seconds and 60° C. for one minute. In orderto quantitatively express the levels of vaccinia virus, a standard curvewas generated using cDNA from purified vaccinia virus.

Statistical Analyses: All statistical analysis was conducted using GraphPad Prism, version 4.01 (San Diego, Calif.). Statistical differences innumber of viral plaques or viral gene expression between multiple groupswas determined using a one-way analysis of variance (ANOVA) andsignificant differences determined by a Tukey-Kramer test (Tukey, J.,Exploratory Data Analysis. Addison Wesley, Reading, N.Y. (1977)).

Observation of CSA Interactions with Vaccinia: A fluorophore labeled CSA(CSA-59) was dissolved in phosphate-buffered saline (PBS) at aconcentration of 1.2 μg/ml. Purified vaccinia (10⁸ PFU/ml) was suspendedin PBS at 10⁴, 10⁵, and 10⁶ PFU/4. An aliquot of the CSA solution wasplaced in a quartz cuvette in a FluoroMax-3 fluorometer. Temperature washeld constant at 25° C. The vaccinia virus was titrated into the CSAsolution, and fluorescence emission was measured (λ_(ex)=340 nm,λ_(ex)=450 nm) in counts per second (CPS). Fluorescent studies withkeratinocytes were performed in an identical manner.

Example 17

This example describes the ability of various CSAs to kill vacciniavirus.

Using a standard viral plaque assay, various CSA were evaluated for theability to kill vaccinia virus (FIG. 11 and Table 1). CSAs 91 and 92were most effective in killing vaccinia virus with significant viralkilling observed with as little as 1 or 5 micromolar concentrations.CSA-7 and CSA-10 were also effective at killing vaccinia virus withsignificant viral killing observed with as little as 5 micromolarconcentration. CSA-8, CSA-13, CSA-17 and CSA-90 were also effective at 1or 5 micromolar concentration. CSA-31 and CSA-54 possessed little or nodetectable activity against vaccinia at the amounts studied.

TABLE 1 %100 %50 %25 %10 %5 %1 Number of % Viability μM μM μM μM μM μMplaques at 0 μM CSA-92 0.00 0.00 0.00 0.00 0.00 0.00 61.67 CSA-91 0.000.00 0.00 0.00 0.00 10.31 74.33 CSA-10 0.00 0.00 0.00 0.00 0.37 91.00CSA-7 0.00 0.00 0.00 0.00 0.56 87.00 CSA-46 0.00 0.00 0.38 3.77 41.8988.33 CSA-13 0.00 0.00 3.31 8.82 37.50 90.67 CSA-17 0.00 0.00 3.00 19.4853.18 89.00 CSA-26 0.00 0.76 4.96 43.13 82.44 87.33 CSA-59 0.00 0.5426.09 66.31 97.83 61.33 CSA-8 1.56 2.33 4.67 28.79 61.87 85.67 CSA-900.48 4.83 7.73 14.49 23.67 64.73 69.00 CSA-21 4.31 14.12 40.39 71.76100.00 85.00 CSA-86 13.62 31.92 40.85 50.70 56.34 72.30 71.00 CSA-2510.04 39.41 66.91 74.35 97.40 89.67 CSA-85 34.84 66.97 84.16 78.28 88.2497.29 73.67 CSA-84 27.05 50.72 50.72 90.82 100.48 100.97 69.00 CSA-8763.94 67.31 83.65 91.59 88.94 86.06 69.33 CSA-89 68.81 76.61 85.78 83.9482.57 88.99 72.67 CSA-15 65.80 97.40 101.49 93.68 98.88 89.67 CSA-1198.36 97.27 100.00 92.90 84.15 88.45 CSA-54 95.53 101.12 95.53 101.12103.91 86.52 CSA-31 101.52 102.28 101.52 99.24 99.24 87.67

Example 18

This example describes data relating to keratinocyte exposure tovaccinia.

Since the virus must use the host cell for survival, human keratinocyteswere infected with vaccinia for 6 hours. At that time, media was removedand replaced with fresh media with or without CSA. Cells and remainingvirus were then allowed to incubate for an additional 18 hours. Cellswere processed for evaluation of vaccinia gene expression by for realtime PCR. In this study, keratinocytes exposed to vaccinia alone hadsignificantly higher levels of vaccinia gene expression than those cellsexposed first to vaccinia and then treated with a CSA. Results indicatethat CSAs demonstrate a range of activity against vaccinia virus. Themost effective compounds are CSA-7, CSA-10, CSA-13, CSA-26 and CSA-46.Considering these antiviral activities and the structures of the CSAstested (FIG. 12), the following conclusions can be drawn:

-   (1) The nature of the group at C24 (see FIG. 10 for steroid    numbering) influences antiviral activity:    -   Charged groups at C24 decreases activity.    -   Addition of a lipid chain at C24 increases activity.-   (2) Loss of a charged group at C7 decreases activity.-   (3) Increasing tether length between the steroid scaffolding and the    amine groups at C3, C7, and C₁₂ decreases activity.-   (4) Dimerization of a CSA results in improved activity.

Example 19

This example describes data showing that CSA-59 preferentiallyincorporates into the vaccinia lipid envelope over the keratinocytemembrane with a high degree of selectivity.

The fluorophore in CSA-59 (FIG. 10) is related to prodan, which is asmall fluorophore that responds to the hydrophobicity of its environmentvia large changes in fluorescence emission wavelength and intensity. Inwater, fluorescence emissions of prodan and PBS-59 are centered at 550nm, and in aprotic solvents the emission intensity increases and iscentered at 450 nm. Studies of interactions of CSA-59 and bacteria havereported that incorporation of the antimicrobial into bacterialmembranes correlated with increased fluorescent intensity at 450 nm.Association of CSA-59 with vaccinia and keratinocytes was compared basedon surface area of the organisms. The surface area of vaccinia wascalculated based on an approximate radius of vaccinia of 300 nm, whilethe surface area of keratinocytes was approximated at 11 μm.

The relationships between surface area and CSA incorporation are shownin FIG. 13. These data indicate that CSA-59 preferentially incorporatesinto the vaccinia lipid envelope over the keratinocyte membrane with ahigh degree of selectivity. With much less surface area of lipidenvelope present, CSA-59 incorporates into the lipid envelope ofvaccinia virus as compared to CSA-59 in the presence of keratinocytes.

1. A method for providing a subject with protection against a poxvirusinfection or pathogenesis, comprising administering a compositioncomprising a sufficient amount of cationic steroid antimicrobial (CSA),CSA-13, to provide the subject with protection against a poxvirusinfection or pathogenesis.
 2. The method of claim 1, wherein the CSA isadministered concurrently with or following contact of the subject withor exposure of the subject to a poxvirus.
 3. The method of claim 1,wherein the CSA is administered concurrently with or followingdevelopment of a symptom or pathology associated with or caused bypoxvirus infection.
 4. The method of claim 1, wherein the poxviruscomprises a pathogenic poxvirus.
 5. The method of claim 4, wherein thepathogenic poxvirus is selected from variola major and variola minorsmallpox virus.
 6. The method of claim 5, wherein the variola major orvariola minor smallpox virus is an extracellular enveloped virus (EEV),intracellular mature virus (IMV) or cell-associated virus (CEV) form. 7.The method of claim 4, wherein the pathogenic poxvirus is selected frommonkeypox, cowpox, Molluscum Contagiosum, camelpox, goatpox, swinepox,sheeppox, buffalopox, sealpox, canarypox, raccoonpox, pigeonpox andrabbitpox.
 8. The method of claim 1, wherein the poxvirus comprises alive or attenuated pathogenic or non-pathogenic Vaccinia virus.
 9. Themethod of claim 8, wherein the Vaccinia virus is selected from vacciniaAnkara (MVA), vaccinia virus Lister strain, vaccinia virus Copenhagenstrain, vaccinia virus Connaught strain, vaccinia virus Brighton strain,vaccinia virus LC16m8strain, vaccinia virus LC16MO, vaccinia virusIHD-J, vaccinia virus Dairen I, vaccinia virus NYCBOH strain, vacciniavirus Wyeth strain, vaccinia virus Tian Tan, vaccinia virus LIVP,vaccinia virus L-IPV, vaccinia virus Dryvax® and ACAM1000.
 10. Themethod of claim 1, wherein the CSA comprises a multimer.
 11. The methodof claim 1, wherein the CSA multimer comprises a dimer, trimer, ortetramer.
 12. The method of claim 1, wherein the CSA comprises apharmaceutically acceptable carrier or excipient.
 13. The method ofclaim 1, wherein the CSA comprises a sterile formulation.
 14. The methodof claim 1, wherein the subject is provided with partial or completeprotection against a poxvirus infection or pathogenesis, or a symptomcaused by a poxvirus infection or pathogenesis.
 15. The method of claim1, wherein the method reduces, decreases, inhibits, ameliorates orprevents onset, severity, duration, progression, frequency orprobability of one or more symptoms associated with a poxvirus infectionor pathogenesis.
 16. The method of claim 15, wherein the symptom isselected from: high fever, muscle aches, fatigue, headache, backache,malaise, rash, blisters, pustules, lesions, delirium, vomiting,diarrhea, excess bleeding and death.
 17. A method for providing asubject with protection against a poxvirus infection or pathogenesis,comprising administering a sufficient amount of CSA-13 to provide thesubject with protection against the poxvirus infection or pathogenesis.18. The method of claim 1, wherein the CSA is administered prior tocontact of the subject with or exposure of the subject to a poxvirus.19. The method of claim 1, wherein the CSA is administered prior todevelopment of a symptom or pathology associated with or caused bypoxvirus infection.